EP3831849A1 - Anticorps bispécifiques contre ceacam5 et cd47 - Google Patents

Anticorps bispécifiques contre ceacam5 et cd47 Download PDF

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EP3831849A1
EP3831849A1 EP19213002.9A EP19213002A EP3831849A1 EP 3831849 A1 EP3831849 A1 EP 3831849A1 EP 19213002 A EP19213002 A EP 19213002A EP 3831849 A1 EP3831849 A1 EP 3831849A1
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seq
light chain
bispecific antibody
cdrl1
cdrl2
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Lamkap Bio Beta Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to bispecific antibodies which bind to human carcinoembryonic antigen CEACAM5 (CEA) and to human CD47 (CEAxCD47 bispecific antibodies) and in one embodiment to human CEACAM5, human CEACAM6 and to human CD47.
  • CEA carcinoembryonic antigen
  • CEAxCD47 bispecific antibodies in one embodiment to human CEACAM5, human CEACAM6 and to human CD47.
  • the present invention relates to polynucleotides encoding such bispecific antibodies and vectors and host cells comprising such polynucleotides.
  • the invention further relates to methods for selecting and producing such antibodies and to methods of using such antibodies in the treatment of diseases.
  • the invention also relates to the therapeutic use of the CEAxCD47 bispecific antibodies in monotherapy and in combination therapy, especially with CEAxCD3 T-cell bispecific antibodies (TCB) and/or inhibitors of PD-1 or PD-L1.
  • TAB T-cell bispecific antibodies
  • CEA human CEA family contains 29 genes, of which 18 are expressed: 7 belonging to the CEA subgroup and 11 to the pregnancy-specific glycoprotein subgroup. Several CEA subgroup members are thought to possess cell adhesion properties. CEA is thought to have a role in innate immunity ( Hammarström S., Semin Cancer Biol. 9(2):67-81 (1999 )).
  • Carcinoembryonic antigen CEA, CEACAM5, Meconium antigen (MA) or CD66e; UniProtKB - P06731
  • CEACAM carcinoembryonic antigen-related cell adhesion molecule
  • Tumoretropericardium a tumor-associated antigen
  • CEACAM6 (CD66c; NCA-50/90; UniProtKB - P40199) belongs also to the carcinoembryonic antigen (CEA) family. Multiple monoclonal antibodies have been raised against CEA for research purposes, as diagnostic tools, and for therapeutic purposes (see e.g. WO2012117002 (incorporated by reference in its entirety), see also Example 8 f)). Soluble CEA - is an established tumor marker. Levels in plasma of cancer patients can go in some cases over 1000 ng/ml, whereas plasma concentrations in healthy individuals are below 10 ng/ml (e.g. Sandler B.
  • the mouse monoclonal antibody PR1A3 was raised by fusion of NS1 (P3/NS 1/I-Ag-4-1) myeloma cells with spleen cells from mice immunized with normal colorectal epithelium Richman P. I. and Bodmer W. F., Int. J. Cancer, 39:317-328, 1987 describe mouse monoclonal antibody PR1A3.
  • Epitope mapping of PR1 A3 shows that the antibody targets the B3 domain and the GPI anchor of the CEA molecule ( Durbin H. et al., Proc. Natl. Scad. Sci. USA, 91 :4313-4317, 1994 ).
  • the PR1A3 antibody binds mainly to the membrane- bound CEA, and not the soluble CEA form that can be found in the bloodstreams of cancer patients.
  • the epitope bound by PR1 A3 is a conformational epitope, not a linear epitope ( Stewart et al., Cancer Immunol Immunother, 47 (1999) 299-06 ).
  • Humanized PR1 A3 (hPR1 A3) antibodies are described e.g. by Conaghhan P. J., et al., Br. J. Cancer, 98 (2008)1217-1225 and WO2012117002 (incorporated by reference in its entirety).
  • CEACAM5 is expressed by the cells of several tumor types including but not limited to e.g. colorectal tumors, tumors of the pancreas, lung tumors, gastric tumors etc.
  • CEACAM6 can be well higher as expression of CEACAM5 (see e.g Blumenthal RD et.al. BMC Cancer, 2007 Jan 3;7:2 ).
  • a method for treating cancer by a combination of a human PD-1 axis antagonist and an anti-CEA/anti-CD3 bispecific antibody is mentioned in US20140242079 and WO2017118657 (each of which is incorporated by reference in its entirety) and clinical results have been published at ASCO conference 2017 ( Tabernero et al, J Clin Oncol 35, 2017 (suppl;abstr 3002)).
  • a method of treating tumors by administering immune checkpoint antagonists binding two or more different targets of an immune checkpoint pathway, and a T cell-redirecting agent binding to CEA and a T cell surface antigen is mentioned in WO2015112534 .
  • a conjugate consisting of a single domain anti-CEACAM6 antibody and urease is at present in clinical trials (NCT02309892; WO2016116907 ).
  • a class I antibody binding to CEACAM5, CEACAM6 and granulocytes is mentioned in US20110064653 .
  • An anti CD3 ⁇ antibody described in the state of the art is SP34 ( Yang SJ, The Journal of Immunology (1986) 137; 1097-1100 ). SP34 reacts with both primate and human CD3. SP34 is available from BD Biosciences.
  • a further anti CD3 antibody described in the state of the art is UCHT-1 (see WO2000041474 ).
  • a further anti CD3 antibody described in the state of the art is BC-3 (Fred Hutchinson Cancer Research Institute; used in Phase I/II trials of GvHD, Anasetti et al., Transplantation 54: 844 (1992 )).
  • SP34 differs from UCHT-1 and BC-3 in that SP-34 recognizes an epitope present on solely the ⁇ chain of CD3 (see Salmeron et al., (1991) J. Immunol. 147: 3047 ) whereas UCHT-1 and BC-3 recognize an epitope contributed by both the ⁇ and ⁇ chains.
  • Anti CD3 antibodies are also described in WO2007042261 , WO2008119565 , WO2008119566 , WO2008119567 , WO2010037836 , WO2010037837 , WO2010037838 , and US8236308 (each of which is incorporated by reference in its entirety).
  • a bispecific antibody comprising a binding part specific for CEA and a binding part specific for CD3 ⁇ is described in US20140242079A1 (incorporated by reference in its entirety).
  • CD47 Human CD47 (UniProtKB - Q08722 (CD47_HUMAN; IAP) is a transmembrane protein that binds the ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRP ⁇ ; CD172a; UniProtKB P78324) and can act as a "don't eat me” signal to the immune system, especially for macrophages.
  • CD47 is involved in a range of cellular processes, including apoptosis, proliferation, adhesion, and migration. Furthermore, it plays a key role in immune and angiogenic responses. CD47 is overexpressed in different tumor cells.
  • Antibodies against CD47 are described in the state of the art and some are in clinical trials as therapeutic agents for tumor treating ( Weiskopf K. European Journal of Cancer 76 (2017) 100-109 ; Huang Y et al., J Thorac Dis 2017;9(2):E168-E174 .
  • Antibodies of the IgG1 subclass that bind CD47 can result in the depletion of platelets and reduction of red blood cells RBC of hemoglobin in a Fc-dependent manner (see e.g. US20140140989 ).
  • WO2014087248 describe a bispecific antibody against CD19 and CD47.
  • a bispecific antibody against CD19 and CD47 comprising a common heavy chain of SEQ ID NO:5 and a variable light domain VL of SEQ ID NO:10 is described in WO2014087248 (incorporated by reference in its entirety).
  • Human FcRI (CD64) is restricted to monocytes/macrophages and dendritic cells (DCs) and, inducibly expressed on neutrophils and mast cells; hFc RIIA (CD32A) is expressed on all myeloid cells but not on lymphocytes; hFc RIIB (CD32B) is highly expressed only on circulating B cells and basophils ( L. Cassard, F. Joensson, S. Arnaud, M. Daeron, J.
  • hFc RIIC CD32C
  • hFc RIIIA CD16A
  • hFcRIIIB CD16B
  • hFc RIIA is the only activating IgG receptor constitutively expressed by mast cells, basophils, neutrophils and eosinophils ( Bruhns P., Blood 119 (2012) 5640 ).
  • the biological activities of each subclass of IgG are poorly known.
  • IgG receptors (Fc ⁇ Rs) are strikingly numerous in humans. They comprise high-affinity and low-affinity receptors. Both high-affinity and low-affinity Fc ⁇ Rs bind IgG-immune complexes with a high avidity, but only high-affinity Fc ⁇ Rs bind monomeric IgG.
  • hFc ⁇ RI high-affinity IgG receptor in humans
  • hFc ⁇ RIIA two families of low-affinity IgG receptors
  • hFc ⁇ RIIA Fc ⁇ R associated activating receptors
  • hFc ⁇ RIIA and hFc ⁇ RIIC Fc ⁇ R associated activating receptors
  • hFc ⁇ RIIB single-domain inhibitory receptors
  • hFc ⁇ RIIIB GPI-anchored receptors whose function is uncertain ( Bruhns P. Blood 113 (2009) 3716 ).
  • an antibody is produced as a population of glycoforms which share the same polypeptide backbone but have different oligosaccharides attached to the glycosylation sites.
  • the oligosaccharides normally found in the Fc region of serum IgG are of complex bi-antennary type ( Wormald et al., Biochemistry 36: 1370-1380 (1997 ), with a low level of terminal sialic acid and bisecting N-acetylglucosamine (GlcNAc), and a variable degree of terminal galactosylation and core fucosylation.
  • Antibodies with a reduced fucose content in glycan moieties exhibit higher antibody dependent cellular cytotoxicity (ADCC) activity compared to a normally fucosylated antibody ( Niwa R et al., Cancer Res, 64, 2127-33, 2004 ).
  • ADCC antibody dependent cellular cytotoxicity
  • the mechanism behind the enhanced ADCC of a low / no-fucose antibody is its increased affinity to Fc ⁇ RIIIa (CD 16).
  • T-cell bispecific antibodies bind to different epitopes of CEAxCD3 and have different tumor cell killing potency.
  • most potent CEAxCD3 T-cell bispecific antibodies described in WO201705389 are by a factor of 10 to 100 or more potent than RO6958688/cibisatamab (CEA-TCB).
  • T-cell bispecific antibodies plus PD-1 axis inhibitors and/or other checkpoint inhibitors or agonists for T-cells.
  • another therapeutic agent aiming to re-direct T-cells against tumor cells of advanced solid tumors, it may be more successful to add a therapeutic agent re-directing to the tumor cells other immune cells, especially macrophages or macrophages and natural killer NK-cells.
  • This invention deals with bispecific antibodies re-directing macrophages and also NK-cells against CEACAM5 or CEACAM5 and CEACAM6 expressing solid tumors as a monotherapy or in combination with e.g. T-cell bispecific antibodies and/or PD-1/PD-L1 inhibiting antibodies.
  • CAR T-cells may have a simple explanation - the number of CAR T-cells penetrating the solid tumor and distributed in it are just not sufficient. This is certainly different in the majority of haematological malignancies; CAR T-cells can well access the tumor cells, explaining the difference of high efficacy in these malignancies compared to disappointing efficacy in solid tumors.
  • CAR T-cells may be heavily suppressed by the tumor microenvironment (TME) which is mostly strongly immune suppressive.
  • TEE tumor microenvironment
  • Monoclonal antibodies and also bispecific antibodies used in therapy can cause a variety of adverse effects.
  • An important toxicity issue is the cytokine-release syndrome (CRS), which was for example found in therapy with alemtuzumab, muromonab-CD3, rituximab, and CD19 x CD3 bispecific antibody blinatumomab.
  • CRS cytokine-release syndrome
  • anti-CD47 antibodies induce increased amounts of pro-inflammatory cytokines after anti-CD47 antibody mediated phagocytosis (see e.g. US20160144009 ).
  • Known adverse events of anti-CD47 monoclonal antibodies with wt IgG1 Fc are increased red blood cell RBC phagocytosis/lysis and platelet activation.
  • the present invention provides bispecific antibodies with a first binding part either binding to human CEACAM5 and not to CEACAM6 or binding to CEACAM 5 and 6 in a balanced manner and a second binding part binding to human CD47.
  • the bispecific antibodies according to the invention do not bind to CEACAM1 to avoid phagocytosis/killing of immune cells because CEACAM1 is widely expressed on immune cells.
  • the bispecific antibodies are designated to the treatment of solid tumors.
  • the bispecific antibodies combine high efficacy with low toxicity, low immunogenicity and favourable pharmacokinetic properties.
  • the bispecific antibodies according to this invention induce their anti-tumor cells effects mainly via optimized phagocytosis/ADCP (antibody dependent cellular phagocytosis) and ADCC (antibody dependent cellular cytotoxicity) due to involvement of immune cells, especially macrophages and NK-cells.
  • the present invention also provides bispecific antibodies specifically binding to human CEACAM5 or human CEACAM5 and 6 and human CD47 designated for the combination treatment with CEAxCD3 T-cell bispecific antibodies like RO6958688, RO7172508 and other CEAxCD3 T-cell bispecific antibodies e.g. as described below and showing strong phagocytosis of tumor cells like MKN-45 in the presence of human macrophages.
  • the present invention provides also monoclonal antibodies specifically binding to human CEACAM5 or human CEACAM5 and CEACAM6 and bispecific antibodies comprising such monoclonal antibodies and their uses, especially for use in the treatment of solid tumors.
  • FIG 1 provides an overview on the general structure of the bispecific antibodies of this invention.
  • the invention relates to a monoclonal antibody (further named also as "AC CEA”) specifically binding to human CEACAM5 (further named also as “CEA) and cynomolgus CEACAM5, characterized in comprising
  • the invention relates to an AC CEA according to the invention, characterized in comprising
  • the invention relates to AC CEA according to the invention, characterized in comprising as heavy chain variable region a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the heavy chain region of SEQ ID NO:5 and as light chain a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain selected from the group of
  • the invention relates to AC CEA according to the invention, characterized in comprising as heavy chain region a heavy chain region of SEQ ID NO:5 and as light chain a light chain selected from the group of
  • the AC CEA is a Fab or a F(ab) 2 fragment. In one embodiment such Fab fragment is the first binding part of a bispecific antibody. In one embodiment the AC CEA is specifically binding to human CEACAM5, cynomolgus CEACAM5 and human CEACAM6.
  • the invention relates to a AC CEA antibody according to the invention, characterized in that said antibody competes for binding to CEACAM5 with the anti-CEACAM5 antibody SM3E, said antibody SM3E comprises as VK and VH domains VK and VH of sequences SEQ ID NO:110 and 111.
  • the invention relates to a AC CEA antibody according to the invention, characterized in that said antibody is a bispecific antibody.
  • the invention relates to an AC CEA antibody according to the invention, characterized in that said antibody is a bispecific antibody comprising as first binding part said AC CEA as monovalent light and heavy chain (VHCH1) and binding in the second binding part specifically to human CD47.
  • VHCH1 monovalent light and heavy chain
  • the invention relates to a bispecific antibody (further named also as “”, “CEAxCD47 bispecific antibody”, or “bispecific antibody according to the invention”) comprising a first binding part specifically binding to human CEACAM5 (further named also as “CEA”) and a second binding part specifically binding to human CD47 (further named also as “CD47”) characterized in that:
  • the invention relates to a bispecific antibody according to the invention, characterized in comprising
  • the invention relates to a bispecific antibody according to the invention, characterized in comprising
  • the invention relates to a bispecific antibody according to the invention, characterized in comprising in the first binding part a heavy chain region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the heavy chain region of SEQ ID NO:5 and as light chain a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain selected from the group of
  • the invention relates to a bispecific antibody according to the invention, characterized in comprising in the first binding part a heavy chain region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the heavy chain region of SEQ ID NO:5 and as light chain a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain selected from the group of
  • the invention relates to a bispecific antibody according to the invention, characterized in comprising in the first binding part a heavy chain region sequence of SEQ ID NO:5 and as light chain a light chain selected from the group of
  • the invention relates to a bispecific antibody according to the invention, characterized in comprising in the first binding part a heavy chain region sequence of SEQ ID NO:5 and as light chain a light chain selected from the group of
  • the invention relates to a bispecific antibody according to the invention, characterized in comprising in the first binding part as variable heavy chain region a variable heavy chain region of SEQ ID NO:4.
  • the invention relates to a bispecific antibody according to the invention, characterized in comprising in the second binding part as variable heavy chain region a variable heavy chain region of SEQ ID NO:4.
  • the invention relates to a bispecific antibody according to the invention, characterized in comprising in the second binding part as variable light chain region a variable light chain region of SEQ ID NO:10.
  • the invention relates to a bispecific antibody according to the invention, characterized in specifically binding to human CEACAM5 and cynomolgus CEACAM5.
  • the invention relates to a bispecific antibody according to the invention, characterized in specifically binding to human CEACAM5, cynomolgus CEACAM5 and human CEACAM6.
  • the invention relates to a bispecific antibody according to the invention, characterized in that said bispecific antibody competes for binding to CEACAM5 with the anti-CEACAM5 antibody SM3E, which comprises as VK and VH domains VK and VH of sequences SEQ ID NO:110 and 111.
  • the bispecific antibody is K2AC53 and K2AC54 and derivatives comprising the CDR regions and/or the light and heavy chains of said antibodies as described above.
  • the invention relates to a bispecific antibody specifically binding to human CEACAM5 and human CD47 characterized in that the Fc region has been glycoengineered to have a reduced number of fucose residues as compared to the same but non-glycoengineered bispecific antibody.
  • the present invention provides a bispecific antibody, characterized in specifically binding to human CEACAM5 and not to human CEACAM6 in the first binding part and specifically binding to human CD47 in the second binding part.
  • the term "not binding to human CEACAM6” means that in an ELISA based binding assay (example 8f) to the recombinant human CEACAM6 protein in one embodiment a concentration dependent binding curve cannot be established due to very weak or no binding or in another embodiment EC50 for binding is 100 or more times higher than EC50 for binding to recombinant human CEACAM5.
  • the present invention provides a bispecific antibody, characterized in specifically binding to human CEACAM5 and human CEACAM6 in the first binding part and to human CD47 in the second binding part.
  • the invention relates to a bispecific antibody CEAxCD47 specifically binding in a balanced manner to human CEACAM5 and human CEACAM6.
  • the bispecific antibody is characterized in binding to human recombinant CEACAM5 and CEACAM6, characterized in that the EC50 values of binding to human CEACAM5 and human CEACAM6 differing by less than a factor of 10.
  • the bispecific antibody is characterized in binding to human CEACAM5 and CEACAM6, characterized in that the EC50 values of binding to human CEACAM5 and human CEACAM6 differing by less than a factor of 5 (balanced CEACAM5 and CEACAM6 binding, binding in balanced manner, see figures 4 and 5 and table 6). Binding is measured in a streptavidin/biotin-based ELISA (see example 8f).
  • Such bispecific antibodies are e.g. K2AC49 and K2AC50 and derivatives comprising the CDR regions and/or the light and heavy chains of said antibodies as described above.
  • the bispecific antibody according to the invention is characterized in specifically binding to human CEACAM5 and human CEACAM6 in the first binding part and human CD47 in the second binding part, characterized in
  • the bispecific antibody according to the invention is characterized in specifically binding to human CEACAM5 and cynomolgus CEACAM5 in the first binding part and human CD47 in the second binding part, characterized in
  • the present invention provides a bispecific antibody, specifically binding to human CEACAM5 and human CEACAM6 in the first binding part and human CD47 in the second binding part, characterized in
  • the present invention provides a bispecific antibody, specifically binding to human CEACAM5 and cynomolgus CEACAM5 in the first binding part and human CD47 in the second binding part, characterized in
  • the antibody is characterized in a balanced human/cynomolgus CEACAM5 binding (EC50 for binding to recombinant human CEACAM5 and cynomolgus CEACAM5 do not differ by more than a factor of 10, in one embodiment a factor of 5). In one embodiment of the invention these antibodies are characterized in human CEACAM6 binding with an EC50 for binding to the recombinant human CEACAM6 (ELISA based assay, see Example 8f) of 1 nM or lower.
  • the present invention provides a bispecific antibody, specifically binding to human CEACAM5 and cynomolgus CEACAM5 in the first binding part and human CD47 in the second binding part, characterized in
  • the invention relates to a bispecific antibody specifically binding to human CEACAM5 and human CD47, the bispecific antibody comprising a first binding part, specifically binding to human CEACAM5 and a second binding part, specifically binding to human CD47, characterized in that said bispecific antibody competes with the anti-CEA antibody SM3E, comprising as VK and VH domains VK and VH of sequences SEQ ID NO:110 and 111, for binding to CEACAM5.
  • the invention relates to a bispecific antibody specifically binding to human CEACAM5 and human CD47, the bispecific antibody comprising a first binding part, specifically binding to human CEACAM5 and a second binding part, specifically binding to human CD47, characterized in that said bispecific antibody does not compete with anti-CEA antibodies SAR, comprising as VK and VH domains VK and VH of sequences SEQ ID NO:112 and 113, and CH1A1A, comprising as VK and VH domains VK and VH of sequences SEQ ID NO:114 and 115 for binding to CEACAM5.
  • SAR anti-CEA antibodies
  • SAR comprising as VK and VH domains VK and VH of sequences SEQ ID NO:112 and 113
  • CH1A1A comprising as VK and VH domains VK and VH of sequences SEQ ID NO:114 and 115 for binding to CEACAM5.
  • the invention relates to a bispecific antibody specifically binding to human CEACAM5 and human CD47, the bispecific antibody comprising a first binding part, specifically binding to human CEACAM5 and a second binding part, specifically binding to human CD47, characterized in that the EC50 value of phagocytosis index curve of said bispecific antibody is in the range of 0.01 to 10 times of the EC50 value of reference antibody K2AC54 under the same experimental conditions. In further embodiments the range is 0.01 to 10, 0.1 to 10, 0.2 to 10, 0.3 to 10, or 0.5 to 10.
  • EC50 values of phagocytosis are measured as EC50 values of the phagocytosis index curve (imaging-based phagocytosis assay, see Example 9 and figure 3 ). In one embodiment the EC50 value is in such range in the presence of 1mg/ml human IgG or without human IgG.
  • the invention relates to a bispecific antibody specifically binding to human CEACAM5 and human CD47, the bispecific antibody comprising a first binding part, specifically binding to human CEACAM5 and a second binding part, specifically binding to human CD47, characterized in that in presence of 1mg/ml human IgG the maximal phagocytosis index (see example 9e; CellInsightTM based assay) of said bispecific antibody is not decreased for 30% or more in comparison to the maximal phagocytosis index measured under the same experimental conditions but without addition of human IgG.
  • the maximal phagocytosis index see example 9e; CellInsightTM based assay
  • the bispecific antibody is characterized in being monovalent for the first binding part and monovalent for the second binding part.
  • the constant and variable framework region sequences are human.
  • the bispecific antibody is characterized in that each of the first and second binding part comprises an immunoglobulin heavy chain and an immunoglobulin light chain. In one embodiment the bispecific antibody is characterized in being of human IgG1 type. In one embodiment the bispecific antibody is a full-length antibody.
  • the bispecific antibody according to the invention is characterized in comprising a first binding part specific for CEA, comprising a lambda light chain variable domain and a lambda light chain constant domain and a second binding part specific for CD47, comprising a kappa light chain variable domain and a kappa light chain constant domain ( ⁇ bispecific antibody, ⁇ Body, type 1; see table 1).
  • the second binding part comprises as light chain LC (CD47 VKCK) the light chain of SEQ ID NO:11 and in the first binding part the constant light chain domain is the lambda light chain constant domain of SEQ ID NO:15.
  • the kappa light chain of SEQ ID NO:11 comprises as variable light chain domain the variable light chain domain of SEQ ID NO:10 (Mab CD47 VK) and as constant light chain domain the constant light chain domain of SEQ ID NO:13 (Mab CD47 CK).
  • the bispecific antibody according to the invention is characterized in comprising a first binding part specifically binding to CEA, comprising a kappa light chain variable domain and a lambda light chain constant domain (hybrid light chain) and a second binding part specifically binding to CD47, comprising a kappa light chain variable domain and a kappa light chain constant domain (hybrid versions of bispecific antibodies K2AC41, K2AC42,K2AC43, K2AC60, K2AC61, K2AC62, K2AC63, K2AC64, K2AC65, and K2AC66; ⁇ bispecific antibody, ⁇ Body, type 2, see table 1 and fig.1B ).
  • the second binding part comprises as kappa light chain (CD47 VKCK) the kappa light chain of SEQ ID NO:11, and in the first binding part the lambda constant light chain domain of SEQ ID NO:15 (AC CEA CL).
  • the kappa light chain of SEQ ID NO:11 comprises as variable light chain domain the variable light chain of SEQ ID NO:10 (CD47 VK) and as constant light chain the constant light chain of SEQ ID NO:13 (CD47 CK).
  • the bispecific antibody according to the invention is characterized in comprising a first binding part specifically binding to CEA, comprising a kappa light chain variable domain and a kappa light chain constant domain and a second binding part specifically binding to CD47, comprising a kappa light chain variable domain and a lambda light chain constant domain (hybrid bispecific antibodies; ⁇ bispecific antibody, ⁇ Body, type 3, fig.1C ).
  • the second binding part comprises as light chain the light chain of SEQ ID NO:181 (MabCD47 VKCL, hybrid light chain) and in the first binding part the kappa CL of SEQ ID NO:16.
  • the light chain of SEQ ID NO:181 (MabCD47 VKCL) comprises as variable light chain domain the kappa variable light chain domain of SEQ ID NO:10 (CD47 VK) and as constant light chain domain the lambda constant light chain domain of SEQ ID NO:14 (CD47 CL).
  • the bispecific antibody according to the invention is of fully human bispecific IgG (especially IgG1) format and in addition a ⁇ bispecific antibody of type 1, type 2 or type 3.
  • the bispecific antibody according to the invention is characterized in being a ⁇ bispecific antibody of type 1, type 2, or type 3 and comprising a common heavy chain (cHC).
  • the common heavy chain comprises as variable heavy chain a variable heavy chain of SEQ ID NO:4.
  • the bispecific antibody according to the invention is characterized in comprising a common heavy chain of SEQ ID NO:5
  • the bispecific antibody is characterized in binding to human CD47 with a binding affinity (K D ) of 100 nM to 600nM, in one embodiment with a binding affinity of 100 nM to 500nM.
  • the bispecific antibody is characterized in binding to MKN-45 cells with an EC50 value of 1 to 250 nM, in one embodiment with an EC50 value of 1 to 200 nM. In one embodiment with an EC50 value of 1 to 150 nM. In one embodiment the bispecific antibody is characterized in binding to MKN-45 cells with a value of 1 to 30 nM. In one embodiment the bispecific antibody is characterized in binding to MKN-45 cells with an EC50 value of 10 to 30 nM. In one embodiment the bispecific antibody is characterized in binding to MKN-45 cells with an EC50 value of 10 to 100 nM.
  • the bispecific antibody is characterized in that it does not cross-react with human CEACAM1.
  • the bispecific antibody according to the invention is characterized in that a bispecific antibody specifically binding to human CEACAM5 and CD3 ⁇ (further named also as CEA-TCB), comprising as heavy chains the heavy chains of SEQ ID NO:107 and 108 and as light chains the light chains of SEQ ID NO: 106 and 109 in a concentration of 300 nM does not shift the EC50 of the binding curve of the bispecific antibody of the invention to MKN-45 cells by more than a factor of 3, in one embodiment towards higher concentrations.
  • the bispecific antibody according to the invention and CEA-TCB are defined as "not competitive” and considered able to bind simultaneously to CEA without significantly interfering in binding to said CEA.
  • CEA-TCB is in clinical trials (cibisatamab or RO 6958688; ClinicalTrials.gov NCT03866239).
  • the bispecific antibody according to the invention is characterized that a bispecific antibody specifically binding to human CEACAM5 and CD3 ⁇ (further named also as CEA-TCB 1), comprising as heavy and light chains the chains of amino acid sequences SEQ ID NO: 102 to 105 in a concentration of 30 nM does not shift the EC50 of the binding curve of the bispecific antibody of the invention to MKN-45 cells by more than a factor of 3, in one embodiment towards higher concentrations.
  • the bispecific antibody according to the invention and CEA-TCB1 are defined as "not competitive” and considered able to bind simultaneously to CEA without significantly interfering in binding to said CEA.
  • the bispecific antibody according to the invention and CEA-TCB and/or CEA-TCB 1 are defined as "not competitive” and considered able to bind simultaneously to CEA without significantly interfering in their binding to said CEA.
  • the bispecific antibody according to the invention is characterized in that a bispecific antibody specifically binding to human CEACAM5 and CD3 ⁇ (further named also as CEA-TCB 1), comprising as heavy and light chains the chains of amino acid sequences SEQ ID NO: 102 to 105, in a concentration of 30 nM does not shift the EC50 of the phagocytosis index curve of the bispecific antibody of the invention to MKN-45 cells by more than a factor of 3, in one embodiment towards higher concentrations.
  • a bispecific antibody specifically binding to human CEACAM5 and CD3 ⁇ (further named also as CEA-TCB 1), comprising as heavy and light chains the chains of amino acid sequences SEQ ID NO: 102 to 105, in a concentration of 30 nM does not shift the EC50 of the phagocytosis index curve of the bispecific antibody of the invention to MKN-45 cells by more than a factor of 3, in one embodiment towards higher concentrations.
  • the bispecific antibody according to the invention and CEA-TCB 1 are defined as "not competitive” and considered able to bind simultaneously to CEA without significantly interfering in their binding to said CEA, and can therefore develop its effect on phagocytosis (CEAxCD47) undisturbed and also its effect on T-cell activation (CEAxTCB1) undisturbed, even if therapeutic levels of both drugs are simultaneously present in the tumor tissue.
  • the bispecific antibody according to the invention is characterized that a bispecific antibody specifically binding to human CEACAM5 and CD3 ⁇ (further named also as CEA-TCB), comprising as heavy and light chains the chains of amino acid sequences SEQ ID NO: 106 to 109 in a concentration of 300 nM does not shift the EC50 of the phagocytosis index curve of the bispecific antibody of the invention to MKN-45 cells by more than a factor of 3, in one embodiment towards higher concentrations.
  • the bispecific antibody according to the invention and CEA-TCB are defined as "not competitive” and considered able to bind simultaneously to CEA without significantly interfering in their binding to said CEA and can therefore develop its effect on phagocytosis (CEAxCD47) undisturbed and also its effect on T-cell activation (CEA-TCB) undisturbed, even if therapeutic levels of both drugs are simultaneously present in the tumor tissue.
  • CEAxCD47 phagocytosis
  • CEA-TCB T-cell activation
  • sequences of SEQ ID NO: 106 to 109 and SEQ ID NO:116 are according to US20140242079 (CEA-TCB) and the sequences of SEQ ID NO:102 to 105 and SEQ ID NO:117 are according to WO2017055389 (CEA-TCB1).
  • CEAxCD47 bispecific antibodies of the invention combined with CEAxCD3 bispecific antibodies like CEA-TCB and CEA-TCB 1 show at least additive or even synergistic % killing of tumor cells in an assay containing e.g. MKN-45 tumor cells and human macrophages and T-cells derived from the same volunteer human donor.
  • the bispecific antibody is characterized in comprising a common heavy chain (cHC) as heavy chain of the first binding part and as heavy chain of the second binding part.
  • cHC common heavy chain
  • the bispecific antibody is characterized in that said common heavy chain of each binding part comprises as CDRs CDRH1 of SEQ ID NO:1, CDRH2 of SEQ ID NO:2 and CDRH3 of SEQ ID NO:3.
  • the bispecific antibody is characterized in that said common heavy chain of each binding part comprises as common variable heavy domain SEQ ID NO:4.
  • the bispecific antibody according to the invention is characterized in inhibiting the interaction between CD47 on MKN-45 cells with an IC50 of 0.1 to 10 nM.
  • SIRP ⁇ SIRP ⁇ , CD172a; UniProtKB P78324
  • IC50 0.1 to 10 nM.
  • the bispecific antibody of the invention is characterized in a concentration dependent phagocytosis (ADCP) of CEACAM5 and/or CEACAM6 expressing tumor cell lines like MKN-45 cells by human macrophages at an EC50 of the bispecific antibody below 40nM, in one embodiment below 10 nM (1 ⁇ g/mL are approx. 6.6 nM).
  • ADCP is measured according to the invention as phagocytosis index (EC50 or maximum) by imaging, usually with an E:T ratio of 1:3 (human macrophages;target cells (tumor cells); see e.g. Fig.3 and table 5 for EC50 values and for max. index of phagocytosis).
  • ADCP can be also measured by Flow Cytometry with an E:T ratio of e.g. 3:1 (human macrophages;target cells (tumor cells). Details of the assay are described in example 9e).2 (Flow cytometry based ADCP assay).
  • the bispecific antibody is characterized in specifically binding to CEACAM5 but is not competing for binding to CEACAM5 on tumor cells like MKN-45 with CEA-TCB and/or CEA-TCB1.
  • the bispecific antibody according to the invention is characterized in that the EC50 value for the binding to MKN-45 cells (EC50 between 1 and 250 nM) is increased by less than a factor of three by addition of CEA-TCB at a concentration of 300 nM respectively by addition of CEA-TCB1 at a concentration of 30 nM (no competition).
  • the CEAxCD47 antibodies of the invention show a 100 or more times higher EC50 for RBC phagocytosis compared to the EC50 measured in the same assay with B6H12.2 (ATCC® HB9771TM; for assay see Example 15).
  • the CEAxCD47 antibodies of the invention do not show significant platelet activation in concentrations up to 200 ⁇ g/mL (see Example 15 for the assay used).
  • the present invention relates to a bispecific antibody according to the invention that has been glycoengineered to have an Fc region with modified oligosaccharides.
  • the bispecific antibody according to the invention comprises a reduced amount of fucose in the oligosaccharide chain(s). It was surprisingly found, that such a glycoengineered bispecific antibody according to the invention is characterized in an at least 3 times lower EC50 value for the phagocytosis index curve measured by the imaging based assay) as the same not glycoengineered (parent) bispecific antibody if measured under the same experimental conditions. In one embodiment EC50 for the phagocytosis index is 5 to 10 times lower, or 10 to 30 times lower). In one embodiment, the Fc region has been modified to have a reduced number of fucose residues as compared to the same but non-glycoengineered bispecific antibody.
  • the Fc region has an increased proportion of bisected oligosaccharides as compared to the non-glycoengineered bispecific antibody.
  • the bisected oligosaccharides are predominantly bisected complex.
  • the glycoengineered antigen binding molecules of the invention have an increased proportion of bisected, nonfucosylated oligosaccharides in the Fc region of said bispecific antibody as compared to the non-glycoengineered bispecific antibody.
  • the bispecific antibodies of the invention may have an increased ratio of GIcNAc residues to fucose residues in the Fc region compared to the non-glycoengineered bispecific antibody.
  • the bisected, nonfucosylated oligosaccharides are predominantly in hybrid form.
  • the bisected, nonfucosylated oligosaccharides are predominantly complex type.
  • the bispecific antibody according to the invention is characterized in that 50% to 100% of the N-linked oligosaccharides in the Fc region are nonfucosylated.
  • the bispecific antibody according to the invention is characterized in that the fucose amount in the oligosaccharide chain(s) of the bispecific antibody according to the invention is reduced by 80% to 100% compared to the fucose content of the respective antibody, if no afucosylation method is applied.
  • the bispecific antibody is characterized in that 50% to 100% of the N-linked oligosaccharides in the Fc region are bisected.
  • the bispecific antibody is characterized that 80% to 100% of the N-linked oligosaccharides in the Fc region are bisected and nonfucosylated.
  • the bispecific antibody is characterized in that concentration/ADCC curve (decrease of EC50 or increase of maximum of ADCC) induced by said glycoengineered antibody is increased by at least a factor of 1.2 compared to the ADCC induced by the same but non-glycoengineered bispecific antibody. In one embodiment ADCC is increased by a factor of 1.2 to 2.0.
  • the bispecific antibody is characterized in an at least 3 times lower EC50 value for the phagocytosis index curve measured by the imaging based assay as compared to the same but not glycoengineered (parent) bispecific antibody if measured under the same experimental conditions.
  • EC50 for the phagocytosis index is 5 to 10 times lower, or 10 to 30 times lower
  • the bispecific antibody is characterized in that the maximal phagocytosis index induced by said glycoengineered antibody and measured by flow cytometry is increased by at least a factor of 1.2 compared to the maximal phagocytosis index induced by the same but non-glycoengineered bispecific antibody. In one embodiment maximal phagocytosis index is increased by a factor of 1.2 to 2.0.
  • the bispecific antibody is characterized in that the maximal phagocytosis index induced by said glycoengineered antibody and measured by imaging is increased by at least a factor of 1.2 compared to maximal phagocytosis index induced by the same but non-glycoengineered bispecific antibody. In one embodiment maximal phagocytosis index is increased by a factor of 1.2 to 2.0.
  • the bispecific antibody according to the invention is characterized in comprising one, two or three amino acid substitutions in the Fc region ("Fc amino acid substitution") selected from the group consisting of mono-substitutions S239D, I332E, G236A, of bi-substitutions I332E and G236A, S239D and I332E, S239D and G236A, and of triple-substitution S329D and I332E and G236A.
  • Fc amino acid substitution selected from the group consisting of mono-substitutions S239D, I332E, G236A, of bi-substitutions I332E and G236A, S239D and I332E, S239D and G236A, and of triple-substitution S329D and I332E and G236A.
  • the bispecific antibody according to the invention is characterized in comprising one, two or three amino acid substitutions in the Fc region selected from the group consisting of mono-substitutions S239D, I332E, G236A, of bi-substitutions I332E and G236A, S239D and I332E, S239D and G236A, and triple-substitution S329D and I332E and G236A and a Fc region which has been glycoengineered to have a reduced number of fucose residues as compared to the same but non-glycoengineered bispecific antibody.
  • the bispecific antibody comprising said substitutions in the Fc region is characterized in that concentration/ADCC curve (decrease of EC50 or increase of maximum of ADCC) induced by said amino acid substituted antibody is increased by at least a factor of 1.2 compared to the ADCC induced by said antibody comprising none of said amino acid substitutions in the Fc region. In one embodiment ADCC is increased by a factor of 1.2 to 2.0.
  • the bispecific antibody comprising said substitutions in the Fc region is characterized in an at least 3 times lower EC50 value for the phagocytosis index curve measured by the imaging based assay as compared to the same (parent) bispecific antibody comprising none of said amino acid substitutions in Fc region, if measured under the same experimental conditions.
  • EC50 for the phagocytosis index is 5 to 10 times lower, or 10 to 30 times lower.
  • the bispecific antibody comprising said substitutions in the Fc region is characterized in that flow cytometry determined maximal phagocytosis (ADCP) induced by said amino acid substituted antibody is increased by at least a factor of 1.2 compared to the ADCP induced by said antibody comprising none of said amino acid substitutions in the Fc region. In one embodiment ADCP is increased by a factor of 1.2 to 2.0. In one embodiment the bispecific antibody comprising said substitutions in the Fc region is characterized in that by imaging determined maximal phagocytosis index induced by said amino acid substituted antibody is increased by at least a factor of 1.2 compared to the ADCP induced by said antibody comprising none of said amino acid substitutions in the Fc region. In one embodiment ADCP is increased by a factor of 1.2 to 2.0.
  • the bispecific antibody according to the invention is characterized in that 50% to 100%, 60% to 100%, 70% to 100% or 80% to 100% of the N-linked oligosaccharides in the Fc region are non-fucosylated. In one embodiment, the bispecific antibody according to the invention is characterized in 50% to 100%, 60% to 100%, 70% to 100% or 80% to 100% of the N-linked oligosaccharides in the Fc region are bisected. In one embodiment, the bispecific antibody according to the invention is characterized in that 50% to 100%, 60% to 100%, 70% to 100% or 80% to 100% of the N-linked oligosaccharides in the Fc region are bisected, nonfucosylated.
  • the glycoengineered bispecific antibody comprises increased effector functions compared to the non-glycoengineered bispecific antibody comprising as common heavy chain SEQ ID NO:6 (common heavy chain of parent bispecific antibody, produced in a CHO K1 cell line CHO-K1 (ATCC® CCL-61TM at standard conditions as defined below).
  • the bispecific antibody according to the invention is characterized in that said glycoengineered bispecific antibody comprises one or more increased effector functions such as those from the group consisting of increased binding affinity to Fc ⁇ Rs, increased binding of macrophages (increased antibody dependent cellular phagocytosis; ADCP), increased binding of NK cells (increased antibody-mediated cellular cytotoxicity; ADCC), and increased binding to monocytes.
  • said glycoengineered bispecific antibody comprises one or more increased effector functions such as those from the group consisting of increased binding affinity to Fc ⁇ Rs, increased binding of macrophages (increased antibody dependent cellular phagocytosis; ADCP), increased binding of NK cells (increased antibody-mediated cellular cytotoxicity; ADCC), and increased binding to monocytes.
  • concentration/phagocytosis index curve measured for the anti-CD47 monoclonal antibody hu5F9-G4 (tested in clinical trials since 2014, see e.g. clinicaltrial.gov) is strongly reduced by the addition of human IgG added in physiological concentrations of 1 mg/mL to the assay (increase of EC50 and decrease of the maximum of the phagocytosis curve measured in imaging based assay).
  • CEAxCD47 antibodies of the invention show only a small shift of a factor of 3 or below of EC50 and 30% decrease or less of the maximum of the concentration/phagocytosis index curve if 1mg/mL human IgG is added compared to same assay but without addition of human IgG.
  • CEAxCD47 antibodies of the invention are characterized in that addition of 1 mg /mL of human IgG to the imaging based phagocytosis assay causes a less than a factor of 0.8 reduction of the maximum of the concentration/phagocytosis index curve and/or a less than a factor of 3 shift of the EC50 towards higher concentrations.
  • a further embodiment of the invention is an isolated polynucleotide characterized in encoding a bispecific antibody according to the invention.
  • a further embodiment of the invention is an expression vector comprising the polynucleotide according to the invention.
  • Appropriate polynucleotides are described in SEQ ID NO:12, 119 -141, and 160 - 166.
  • a further embodiment of the invention is a host cell comprising the expression vector according to the invention.
  • a further embodiment of the invention is a method for the production of a bispecific antibody according to the invention, characterized in comprising:
  • the invention is characterized in comprising a method for producing a glycoengineered bispecific antibody according to the invention, lacking fucose or with a reduced amount of fucose on its oligosaccharide chains in a host cell, said method comprising:
  • the second polypeptide encoding the antibody of the invention can be one polypeptide encoding all respective two different light chains and the common heavy chain or separate polypeptides, encoding separately the respective light and heavy chains.
  • the expression vector can be one, two or three vectors expressing the respective two different light chains and the common heavy chain.
  • the invention is characterized in comprising a method for producing a glycoengineered bispecific antibody according to the invention in a host cell, said method comprising:
  • the invention is characterized in comprising a method for producing a glycoengineered bispecific antibody in a host cell, said method comprising:
  • the invention is characterized in comprising a method for producing a Fc substituted bispecific antibody according to the invention in a host cell, said method comprising:
  • a further embodiment of the invention is a method of inducing cell lysis of a tumor cell comprising contacting the tumor cell with a bispecific antibody according to the invention.
  • the tumor cell is a human tumor cell, preferably in a patient.
  • a further embodiment of the invention is a method according to the invention, characterized in that the tumor cell is a colorectal cancer cell, NSCLC (non-small cell lung cancer) cell, gastric cancer cell, pancreatic cancer cell, breast cancer cell, or another tumor cell expressing CEACAM5 or CEACAM5 and CEACAM6.
  • NSCLC non-small cell lung cancer
  • a further embodiment of the invention is a method of treating a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody according to the invention.
  • a further embodiment of the invention is a method of increasing survival time in a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6, said method comprising administering to said subject a therapeutically effective amount of a bispecific antibody according to the invention.
  • a further embodiment of the invention is a method according to the invention, characterized in that the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer or breast.
  • NSCLC non-small cell lung cancer
  • gastric cancer pancreatic cancer or breast.
  • a further embodiment of the invention is a method according to the invention, characterized in that a bispecific antibody according to the invention is administered in combination with chemotherapy or radiation therapy to a human subject.
  • a further embodiment of the invention is a method of treating a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody according to the invention, characterized in that the EC50 value of phagocytosis of said bispecific antibody is in the range of 0.01 to 10 times of the E50 value of reference antibody K2AC54 under the same experimental conditions and in the presence and/or without of 1mg/ml human IgG. In further embodiments the range is 0.01 to 10, 0.1 to 10, 0.2 to 10, 0.3 to 10, or 0.5 to 10.
  • bispecific antibody is characterized in binding to human CD47 with a binding affinity of 100 nM to 600nM, in one embodiment with a binding affinity of 100 nM to 500nM.
  • Bispecific antibody K2AC54 is described by sequences SEQ ID NO:1 to 13, 15, 53, 54, 55 and 89.
  • a further embodiment of the invention is the use of a bispecific antibody according to the invention in a method of treating a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody according to the invention, characterized in that the EC50 value of phagocytosis of said bispecific antibody is in the range of 0.01 to 10 times of the E50 value of reference antibody K2AC54 under the same experimental conditions and in the presence or without of 1mg/ml human IgG. In further embodiments the range is 0.01 to 10, 0.1 to 10, 0.2 to 10, 0.3 to 10, 0.5 to 10 or 0. In one embodiment the bispecific antibody is characterized in binding to human CD47 with a binding affinity of 100 nM to 600nM, in one embodiment with a binding affinity of 100 nM to 500nM.
  • ADCC and ADCP/phagocytosis index values of antibodies according to the invention are not or only to a low extend affected by human IgG in a concentration of 1 mg/ml (1 mg/ml or even higher human IgG is the magnitude of the concentration of IgG found in the blood respectively plasma of most patients), whereas for an anti-CD47 antibody of the state of the art (hu5F9-G4), ADCC and ADCP values are strongly reduced in the presence of 1 mg/mL human IgG.
  • a further embodiment of the invention is the use of the bispecific antibody according to the invention in the manufacture of a medicament for treating a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6.
  • a further embodiment of the invention is the use of the bispecific antibody according to the invention in the manufacture of a medicament according to the invention, characterized in that the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer and breast cancer.
  • NSCLC non-small cell lung cancer
  • gastric cancer pancreatic cancer
  • breast cancer breast cancer
  • a further embodiment of the invention is a bispecific antibody according to the invention, for use in simultaneous, separate, or sequential combination with a second bispecific antibody comprising a third binding part specifically binding to human CEACAM5, and a fourth binding part specifically binding to human CD3 ⁇ in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6.
  • a further embodiment of the invention is a bispecific antibody according to the invention, for use in simultaneous, separate, or sequential combination with a second bispecific antibody comprising a third binding part specifically binding to human CEACAM5 and a fourth binding part specifically binding to an epitope of human CD3 ⁇ , said epitope comprising the amino acid sequence of SEQ ID NO:118 in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6.
  • a further embodiment of the invention is a bispecific antibody according to the invention, for use in simultaneous, separate, or sequential combination with CEA-TCB or CEA/TCB1 in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6.
  • a further embodiment of the invention is a bispecific antibody according to the invention, characterized in not competing with said second bispecific antibody for use in simultaneous, separate, or sequential combination with said second bispecific antibody in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6.
  • a further embodiment of the invention is a bispecific antibody according to the invention, characterized in not competing with CEA-TCB or CEA-TCB 1 for use in simultaneous, separate, or sequential combination with said CEA-TCB or CEA-TCB 1 in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6.
  • a further embodiment of the invention is a bispecific antibody according to the invention, characterized in competing with CEA-TCB or CEA-TCB1 for use in simultaneous, separate, or sequential combination with said CEA-TCB or CEA-TCB 1 in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6.
  • a further embodiment of the invention is a bispecific antibody according to the invention, for use in simultaneous, separate, or sequential combination with a second bispecific antibody comprising a third binding part specifically binding to human CEACAM5, comprising as heavy chain region a heavy chain variable region of SEQ ID NO:98 and as light chain variable region a light chain variable region of SEQ ID NO:99 and a fourth binding part specifically binding to human CD3 ⁇ , comprising as heavy chain variable region a heavy chain variable region of SEQ ID NO:100 and as light chain variable region a light chain variable region of SEQ ID NO:101.
  • a further embodiment of the invention is a bispecific antibody according to the invention, for use according to the invention, characterized in that the bispecific antibody according to the invention and the second bispecific antibody are administered to said subject alternately in 6 to 15 day intervals.
  • a further embodiment of the invention is a bispecific antibody according to the invention, for use according to the invention, characterized in that the bispecific antibody according to the invention and the second bispecific antibody are administered to said subject simultaneously in 6 to 15 day intervals.
  • a further embodiment of the invention is a first bispecific antibody according to the invention, comprising a first binding part, specifically binding to human CEACAM5 and a second binding part, specifically binding to human CD47, for use in simultaneous, separate, or sequential combination in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6, with a second bispecific antibody, comprising a third binding part specifically binding to human CEACAM5, comprising as as heavy chain variable region heavy chain variable region of SEQ ID NO:116 and as light chain variable region a light chain variable region of SEQ ID NO:117 and a fourth binding part specifically binding to an epitope of human CD3 ⁇ , comprising the amino acid sequence of SEQ ID NO:118, whereby said second bispecific antibody in a concentration of 300 nM does not shift the EC50 value of the phagocytosis index curve to MKN-45 cells of the bispecific antibody according to the invention by more than a factor of 3, in one embodiment towards higher concentrations.
  • a further embodiment of the invention is a first bispecific antibody according to the invention, comprising a first binding part, specifically binding to human CEACAM5 and a second binding part, specifically binding to human CD47, for use in simultaneous, separate, or sequential combination in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6, with a second bispecific antibody comprising a third binding part specifically binding to human CEACAM5, comprising as heavy chain variable region a heavy chain variable region of SEQ ID NO:108 and as light chain variable region a light chain variable region of SEQ ID NO:109 and a fourth binding part specifically binding to human CD3 ⁇ , comprising as heavy chain variable region a heavy chain variable region of SEQ ID NO:100 and as light chain variable region a light chain variable region of SEQ ID NO:101, whereby said second bispecific antibody in a concentration of 30 nM does not shift the EC50 of the binding curve to MKN-45 cells of the bispecific antibody according to the invention by more than a factor of 3, in
  • a further embodiment of the invention is a first bispecific antibody according to the invention, for use in simultaneous, separate, or sequential combination in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6, with CEA-TCB or CEA-TCB1, whereby said CEA-TCB in a concentration of 300 nM or CEA-TCB1in a concentration of 30 nM do not shift the EC50 of the binding curve to MKN-45 cells of the bispecific antibody according to the invention by more than a factor of 3, in one embodiment towards higher concentrations.
  • a further embodiment of the invention is a first bispecific antibody according to the invention, comprising a first binding part, specifically binding to human CEACAM5 and a second binding part, specifically binding to human CD47 according to the invention, for use according to the invention, characterized in that said cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer and breast cancer.
  • NSCLC non-small cell lung cancer
  • a further embodiment of the invention is a composition comprising a bispecific antibody according to the invention, characterized in not competing with said second bispecific antibody as defined above for use in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6.
  • a further embodiment of the invention is a composition comprising a bispecific antibody according to the invention, characterized in not competing with a second bispecific antibody comprising a third binding part specifically binding to human CEACAM5, comprising as heavy chain variable region a heavy chain variable region of SEQ ID NO:116 and as light chain variable region a light chain variable region of SEQ ID NO:117 and a fourth binding part specifically binding to an epitope of human CD3 ⁇ , comprising the amino acid sequence of SEQ ID NO:118, for use in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6.
  • a further embodiment of the invention is a composition comprising a bispecific antibody according to the invention, characterized in not competing with a second bispecific antibody comprising a third binding part specifically binding to human CEACAM5, comprising as heavy chain variable region a heavy chain variable region of SEQ ID NO:108 and as light chain variable region a light chain variable region of SEQ ID NO:109 and a fourth binding part specifically binding to human CD3 ⁇ , comprising as heavy chain variable region a heavy chain variable region of SEQ ID NO:100 and as light chain variable region a light chain variable region of SEQ ID NO:101, for use in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6.
  • a further embodiment of the invention is a composition comprising a bispecific antibody according to the invention, characterized in not competing with CEA-TCB and/or CEA-TCB 1.
  • a further embodiment of the invention is a method for the treatment of a human patient diagnosed with a tumor (cancer), especially a solid tumor, especially a solid cancer that expresses CEACAM5 or CEACAM5 and CEACAM6, especially colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer and breast cancer, comprising administering an effective amount of an bispecific antibody according to the invention and a second bispecific antibody as described above, against CEA and CD3 (in one embodiment CEA-TCB or CEA-TCB 1), to the human patient, the method comprising subsequently:
  • the two bispecific antibodies can also be administered in a manner ("simultaneous manner") that the patient experiences therapeutically effective plasma and tissue concentrations of both bispecific antibodies in parallel, e.g.
  • a dose of 0.1 to 10 mg/kg in a further embodiment of 0.5 to 10 mg/kg, in a further embodiment of 1 to 2 mg/kg of the CEA x CD3 bispecific antibody and 1 to 20 mg/kg of the CEA x CD47 bispecific antibody of this invention, followed by one or more of these combined administrations at a frequency of q1w or q2w or q3w or optionally q4w.
  • q1w means administration once a week; q2w means administration every two weeks etc.
  • q1w means administration once a week; q2w means administration every two weeks etc.
  • a further embodiment of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody according to the invention and a pharmaceutically acceptable excipient or carrier.
  • a further preferred embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention for use as a medicament.
  • a further preferred embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention for use as a medicament in the treatment of solid tumor disorders.
  • a further preferred embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention for use as a medicament in the treatment of colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer or breast cancer.
  • a further embodiment of the invention is a composition comprising a bispecific antibody according to the invention, for use in simultaneous, separate, or sequential combination in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6, with a second bispecific antibody, comprising a third binding part specifically binding to human CEACAM5, comprising as heavy chain variable region a heavy chain variable region of SEQ ID NO:116 and as light chain variable region a light chain variable region of SEQ ID NO:117 and a fourth binding part specifically binding to an epitope of human CD3 ⁇ , comprising the amino acid sequence of SEQ ID NO:118, whereby said second bispecific antibody in a concentration of 300 nM does not shift the EC50 of the binding curve to MKN-45 cells of the bispecific antibody according to the invention by more than a factor of 3, in one embodiment towards higher concentrations.
  • a further embodiment of the invention is a composition comprising a bispecific antibody according to the invention, for use in simultaneous, separate, or sequential combination in the treatment of a subject having a cancer that expresses CEACAM5 or CEACAM5 and CEACAM6, with a second bispecific antibody comprising a third binding part specifically binding to human CEACAM5, comprising as heavy chain variable region a heavy chain variable region of SEQ ID NO:108 and as light chain variable region a light chain variable region of SEQ ID NO:109 and a fourth binding part specifically binding to human CD3 ⁇ , comprising as heavy chain variable region a heavy chain variable region of SEQ ID NO:100 and as light chain variable region a light chain variable region of SEQ ID NO:101, whereby said second bispecific antibody in a concentration of 30 nM does not shift the EC50 of the binding curve to MKN-45 cells of the bispecific antibody according to the invention by more than a factor of 3, towards higher concentrations.
  • a further embodiment of the invention is a composition according to the invention, characterized in that the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, or breast cancer.
  • NSCLC non-small cell lung cancer
  • gastric cancer gastric cancer
  • pancreatic cancer pancreatic cancer
  • breast cancer breast cancer
  • a further embodiment of the invention is the use of an antibody according to the invention for the manufacture of a pharmaceutical composition.
  • a further embodiment of the invention is the use of an antibody according to the invention and a pharmaceutically acceptable excipient or carrier for the manufacture of a pharmaceutical composition.
  • a further embodiment of the invention is the use of an antibody according to the invention for the manufacture of a medicament in the treatment of solid tumor disorders.
  • a further embodiment of the invention is the use of an antibody according to the invention in the treatment of colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer or breast cancer.
  • Another aspect of the invention provides a method of inducing cell lysis of a tumor cell comprising contacting the tumor cell with the bispecific antibody of any of above described embodiments.
  • the tumor cell is a colorectal cancer cell, NSCLC (non-small cell lung cancer), gastric cancer cell, pancreatic cancer cell or breast cancer cell.
  • the cell lysis is induced by antibody dependent cellular phagocytosis and/or antibody dependent cellular cytotoxicity of the bispecific antibody.
  • Another aspect of the invention provides a method of treating a subject having a cancer that abnormally expresses CEACAM5 or CEACAM5 and CEACAM6, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody of any of above described embodiments.
  • Another aspect of the invention provides a method of treating a subject having a cancer that abnormally expresses CEACAM5 or CEACAM5 and CEACAM6, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody of any of above described embodiments in combination with a bispecific antibody binding to human CEA and human CD3.
  • CEAxCD3 bispecific antibodies and the CEAxCD47 bispecific antibodies according to the invention are not or only minimally competing they can be not only given sequentially but also in parallel (simultaneously) which may well be an advantage because tumor cell killing via engagement of T-cells by the CEAxCD3 bispecific antibody and at the same time via engagement of macrophages by the CEAxCD47 bispecific antibody is additive or may be even synergistic, which means efficacy is increased if both drugs are given in parallel.
  • Another aspect of the invention provides a method of increasing progression free survival and/or overall survival time in a subject having a cancer that abnormally expresses CEACAM5 or CEACAM5 and CEACAM6, said method comprising administering to said subject a therapeutically effective amount of the bispecific antibody of any of above described embodiments.
  • the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer or breast cancer or another cancer expressing CEACAM5 or CEACAM5 and CEACAM6.
  • the bispecific antibody is administered in combination with chemotherapy or radiation therapy.
  • the subject is a patient suffering from colorectal cancer or lung cancer or gastric cancer or pancreatic cancer or breast cancer or another cancer expressing CEACAM5 or CEACAM5 and CEACAM6.
  • Another aspect of the invention provides a method of treating a subject having a cancer that abnormally expresses CEACAM5 or CEACAM5 and CEACAM6, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody of any of above described embodiments in combination with a bispecific antibody against human CEA and human CD3epsilon.
  • Another aspect of the invention provides a method of increasing progression free survival time and/or overall survival time in a subject having a cancer that abnormally expresses CEACAM5 or CEACAM5 and CEACAM6, said method comprising administering to said subject a therapeutically effective amount of the bispecific antibody of any of above described embodiments.
  • the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer or breast cancer.
  • the bispecific antibody is administered in combination with chemotherapy or radiation therapy.
  • the subject is a cancer patient with colorectal cancer or lung cancer or gastric cancer or pancreatic cancer or breast cancer or another CEACAM5 or CEACAM5 and CEACAM6 expressing cancer.
  • the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer and breast cancer.
  • NSCLC non-small cell lung cancer
  • gastric cancer gastric cancer
  • pancreatic cancer breast cancer
  • antigenic determinant such as CEA, CD47 and CD3.
  • a binding part that binds membrane-bound human carcinoembryonic antigen (CEA, same as CEACAM5) or to CD47 specifically binds to CEA or CD47, more particularly to cell surface or membrane-bound CEA or CD47. Therefore, each binding part binds either to CEA or CD47.
  • CEA membrane-bound human carcinoembryonic antigen
  • each binding part binds either to CEA or CD47.
  • specifically binding, specific for, binding to is meant that the binding is selective for the antigen and can be discriminated from unwanted or nonspecific interactions.
  • the extent of binding of an anti-target antibody to an unrelated, non-target protein is about 10-fold preferably >1 00-fold less than the binding of the antibody to said target as measured, e.g., by surface plasmon resonance (SPR) e.g.
  • SPR surface plasmon resonance
  • Biacore® enzyme-linked immunosorbent (ELISA) or flow cytometry (FACS).
  • Targets are the proteins discussed herein - e.g. CEA, CD47, and CD3 ⁇ .
  • the CEA binding part binds in addition to CEACAM6.
  • "Specifically binding to CEA, CD47, binding to CEA, CD47, specific for CEA, CD47” refers in one embodiment to an antibody, e.g., bispecific antibody, that is capable of binding to the targets CEA and. CD47 with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting tumor cells expressing CEACAM5 or CEACAM5 and CEACAM6 and CD47.
  • binding to MKN-45 cells with an EC50 value of refers to an EC50 value measured by FACS/flow cytometry (see example 10a).
  • the bispecific antibody according to the invention binds to cynomolgus CEACAM5 as well as human CEACAM5.
  • the bispecific antibody is characterized in binding to human CEACAM5 and cynomolgus CEACAM5 with an EC50 ratio of 1:10 to 10:1, in one embodiment 1:5 to 5:1, or in one embodiment 1:3 to 3:1.
  • the term “antibody” refers to an antibody comprising two heavy chains and two light chains. In one embodiment the antibody is a full-length antibody.
  • antibody heavy chain refers to an antibody heavy chain, consisting of a variable region and a constant region as defined for a full-length antibody.
  • antibody light chain refers to an antibody light chain, consisting of a variable region and a constant region as defined for a full-length antibody.
  • full-length antibody denotes an antibody consisting of two “full-length antibody heavy chains” and two “full-length antibody light chains”.
  • a “full-length antibody heavy chain” is a polypeptide consisting in N-terminal to C- terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH-HR-CH2-CH3.
  • a “full-length antibody light chain” is a polypeptide consisting in N-terminal to C- terminal direction of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), abbreviated as VL-CL.
  • the antibody light chain constant domain (CL) can be ⁇ (kappa) or ⁇ (lambda).
  • the two full-length antibody domains are linked together via inter-polypeptide disulphide bonds between the CL domain and the CH1 domain and between the hinge regions of the full-length antibody heavy chains.
  • Examples of typical full-length antibodies are natural antibodies like IgG (e.g. IgG 1 and IgG2), IgM, IgA, IgD, and IgE.
  • the full-length antibody according to the invention is in one embodiment of human IgG1 type, in one further embodiment comprising one or more amino acid substitutions in the Fc part as defined below and/or being glycoengineered at polysaccharide chain attached to Asn297.
  • the full-length antibody according to the invention comprise two binding parts each formed by a pair of VH and VL, one binding to CEA and the other binding to CD47.
  • hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence ("complementarity determining regions” or “CDRs") and/or form structurally defined loops ("hypervariable loops") and/or contain the antigen-contacting residues ("antigen contacts").
  • HVRs are non-contiguous antigen combining sites (also known as antigen binding regions).
  • antibodies comprise six HVRs: three in the VH (HVRH1, HVRHH2, HVRH3), and three in the VL (HVRL1, HVRL2, HVRL3).
  • Exemplary HVRs herein include:
  • CDR Complementarity determining region(s)
  • HVRs hypervariable regions
  • CDRL1 of SEQ ID NO:x refers to that the CDRL1 region of the referred variable light chain is of SEQ ID NO:x (comprising as CDRL1 a CDRL1 of SEQ ID NO:x). This is true also for the other CDRs.
  • HVR residues are numbered herein according to Kabat et al., supra and named as "CDRs”.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in an antibody sequence that are identical with the amino acid residues in the reference antibody sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix.
  • the FASTA program package was authored by W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448 ; W. R. Pearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227-258 ; and Pearson et. al. (1997) Genomics 46:24-36 and is publicly available from http://fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml.
  • the term "90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity" as used in an embodiment of the invention means that in this embodiment all such sequences are either of 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity, or 100% identity (fully identical).
  • Fc region refers to a C-terminal region of an IgG heavy chain; in case of an IgG1 antibody, the C-terminal region comprises -CH2-CH3 (see above).
  • the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226 to the carboxyl-terminus.
  • Constant regions are well known in the state of the art and e.g. described by Kabat, E.A., (see e.g. Johnson, G., and Wu, T.T., Nucleic Acids Res.28 (2000) 214-218 ; Kabat, E.A., et al, Proc. Natl. Acad. Sci. USA 72 (1975) 2785- 2788 ).
  • epitope includes any polypeptide determinant capable of specific binding to an antibody.
  • epitope includes chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics.
  • An epitope is a region of a target that is bound by an antibody.
  • the bispecific antibody of the invention binds to the N-terminal domain of CEACAM5 (Ig-like V-type domain of amino acids 35 - 144, UniProtKB - P06731). Binding location of the CEAxCD47 bispecific antibodies to CEACAM5 is achieved via epitope binning.
  • antibodies are tested in a pairwise combinatorial manner, and antibodies that compete for the same binding region are grouped together into bins. Competition testing is performed herein with anti-CEA antibodies according to the state of the art and as described herein.
  • the bispecific antibody of the invention competes for binding to CEACAM5 with reference antibody SM3E (bin 1). In one embodiment the bispecific antibody of the invention does not compete for binding to CEACAM5 with reference antibodies SAR (bin 2).
  • Competition is measured by an assay wherein biotinylated human CEACAM5 in a concentration of 0.5 ⁇ g/ml is immobilized and incubated with 10 ⁇ g/ml of the reference.
  • CEACAM5 antibodies comprising the CEACAM5 binding part of the CEAxCD47 bispecific antibody of the present invention are added at 0.2 ⁇ g/ml for 1 hour at room temperature. The plate is washed and the bound CEACAM5 mAbs are detected.
  • a common heavy chain refers to a polypeptide consisting in N-terminal to C-terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH-HR-CH2-CH3.
  • VH antibody heavy chain variable domain
  • CH1 antibody constant heavy chain domain 1
  • HR antibody hinge region
  • CH2 antibody heavy chain constant domain 2
  • CH3 antibody heavy chain constant domain 3
  • Common heavy chains suitable for the bispecific antibodies according to the invention are heavy chains of an anti-CD47 antibody as described in WO2012023053 , WO2013088259 , WO2014087248 , and WO2016156537 (each of which is incorporated by reference in its entirety).
  • common heavy chain of the bispecific antibody according to the invention comprises as heavy chain CDRs a CDRH1 of SEQ ID NO:1, a CDRH2 of SEQ ID NO:2 and a CDRH3 of SEQ ID NO:3.
  • the cHC of the bispecific antibody according to the invention comprises as heavy chain variable region VH a VH region of SEQ ID NO:4.
  • the common heavy chain cHC of the bispecific antibody according to the invention is of SEQ ID NO:5 (VH-CH1).
  • the common heavy chain cHC of the bispecific antibody according to the invention is of SEQ ID NO:6.
  • SEQ ID NO:6 is a heavy chain comprising in addition an IgG1 Fc part.
  • the antibody according to the invention is a ⁇ bispecific antibody comprising a cHC ( ⁇ Body).
  • the ⁇ Body format allows the affinity purification of bispecific antibodies which are undistinguishable from a standard IgG molecule and with characteristics that are undistinguishable from a standard monoclonal antibody (see e.g. WO2013088259 , WO2012023053 ), promising no or low immunogenicity potential in patients.
  • Bispecific antibodies of the invention comprising a common heavy chain
  • WO2012023053 incorporated by reference in its entirety
  • the methods described in WO2012023053 generate bispecific antibodies that are identical in structure to a human immunoglobulin.
  • This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant Kappa domain and second light chain variable region fused to a constant Lambda domain.
  • One binding site displays specificity to CEA and the other site displays specificity to CD47, wherein to each the heavy and the respective light chain contribute.
  • the light chain variable regions can be of the Lambda or Kappa family and are preferably fused to a Lambda and Kappa constant domains, respectively.
  • bispecific antibodies of the invention by fusing a Kappa light chain variable domain to a constant Lambda domain for a first specificity or fusing a Lambda light chain variable domain to a constant Kappa domain for the second specificity.
  • the other light chain is then always fully kappa (VL and CL) or fully lambda.
  • the bispecific antibodies described in WO 2012023053 are " ⁇ Bodies".
  • This ⁇ -Body format allows the affinity purification of a bispecific antibody that is undistinguishable from a standard IgG molecule with characteristics that are undistinguishable from a standard monoclonal antibody and, therefore, favourable as compared to previous formats including e.g. amino acid bridges or other unnatural elements.
  • the formats of the bispecific antibodies of the invention are shown in table 1A and figures 1A , B, C).
  • Table 1A (see also fig.lA, B, C; K2AC54, K2AC41, AC41K2 H-CL1 can be taken as exemplary formats)
  • CEACAM CD47 bsAb CEACAM variable light chain
  • CEACAM constant light chain CD47 variable light chain CD47 constant light chain
  • Non-hybrid format Hybrid format in binding part K2AC41 kappa lambda kappa kappa CEACAM5 AC41K2 kappa kappa kappa lambda CD47 H-CL1 K2AC42 kappa lambda kappa kappa CEACAM5 AC42K2 kappa kappa kappa lambda CD47 H-CL1
  • bispecific antibodies according to the invention bind all to bin 1, i.e. distal of the cell membrane.
  • Table 1B CEACAM CD47 bsAb CEACAM variable light chain CEACAM constant light chain CD47 variable light chain CD47 constant light chain K2AC58 lambda lambda kappa kappa K2AC59 lambda lambda kappa kappa K2AC21 lambda lambda kappa kappa kappa
  • K2AC21 comprises in the first binding part as light chain a light chain having of SEQ ID NO:185, and comprises as a CDRL1 of SEQ ID NO:182, CDRL2 of SEQ ID NO:183, CDRL3 of SEQ ID NO:184, in the second binding part as light chain a light chain of SEQ ID NO:11 and as heavy chain a common heavy chain of SEQ ID NO:4.
  • K2AC58 comprises in the first binding part as light chain a light chain having of SEQ ID NO:93, and comprises as a CDRL1 of SEQ ID NO:65, CDRL2 of SEQ ID NO:66, CDRL3 of SEQ ID NO:67, in the second binding part as light chain a light chain of SEQ ID NO:11 and as heavy chain a common heavy chain of SEQ ID NO:4.
  • K2AC59 comprises in the first binding part as light chain a light chain having of SEQ ID NO:94, and comprises as a CDRL1 of SEQ ID NO:68, CDRL2 of SEQ ID NO:69, CDRL3 of SEQ ID NO:70, in the second binding part as light chain a light chain of SEQ ID NO:11 and as heavy chain a common heavy chain of SEQ ID NO:4.
  • Phagocytosis achieved with K2AC21, K2AC58 and K2AC59 is surprisingly strongly inferior to phagocytosis achieved with e.g. reference biAb K2AC54 or other antibodies of the invention binding to bin 1 (see e.g. figure 7 and 3 ). Binding closer to the membrane seems to be a disadvantage compared to binding distal to the membrane.
  • the term “K2ACxx” refers to a bispecific CEACAM5xCD47 antibody according to the invention as defined by the light and heavy chain CDRs. In one embodiment, the term “K2ACxx” refers to a bispecific CEACAM5xCD47 antibody according to the invention as defined by the light and heavy chain CDRs and light and chains which are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the respective light chains. In one embodiment, the term “K2ACxx” refers to a bispecific CEACAM5xCD47 antibody according to the invention as defined by the light and heavy chain CDRs and the respective light chains.
  • K2ACxx refers to a bispecific CEACAM5xCD47 antibody according to the invention as defined by the light and heavy chain CDRs, and the respective light and heavy chains.
  • the respective combinations of CDRs, light and heavy chains can be seen from tables 1 and 2.
  • CEA CEACAM5
  • CEA human carcinoembryonic antigen
  • CD66e CD66e
  • UniProtKB - P06731 a cell surface glycoprotein and a tumor-associated antigen
  • CEACAM6 refers to human CEACAM6 (CD66c; UniProtKB - P40199), which is also a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family.
  • CEACAM5 relates to macaca fascicularis CEACAM5 (NCBI Reference Sequence: XP_005589491.1).
  • CEACAM1 refers to human CEACAM1 (UniProtKB - P13688 (CEAM1_HUMAN) which is also a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Further information and information on other members of the CEA family can be found under http://www.uniprot.org.
  • the bispecific antibody according to the invention is not competitive with CEA-TCB, or CEA-TCB1.
  • a bispecific anti-CEA x anti-CD3 ⁇ antibody (CEA-TCB) is described in Bacac et al Clin. Cancer Res., 22(13), 3286-97 (2016 ) and the variable chainsof CEA-TCB are described in US20140242079 (SEQ ID NO:21 and 27 of US20140242079 (incorporated by reference in its entirety).
  • bispecific CEAxCD3 Mab (CEA-TCB1) is described in WO2017055389 as molecule B "2+1 IgG CrossFab, inverted” with charge modifications (VH/VL exchange in CD3 binder, charge modification in CEA binder, humanized CEA binder) (see SEQ ID NOs 34, 36-38 of WO2017055389 (incorporated by reference in its entirety)).
  • bispecific CEA x CD3 antibody refers to antibody CEA-TCB or antibody CEA-TCB 1.
  • CD47 specifically binding to CD47, binding to CD47, CD47 binding part
  • Human CD47 is a multi-pass membrane protein and comprises three extracellular domains (amino acids 19-141, 198-207, and 257-268; see UniProtKB - Q08722).
  • binding affinity to CD47 is measured quantitatively by SPR.
  • binding of the bispecific antibody according to the invention to CD47 occurs via one or more of said extracellular domains.
  • the bispecific antibodies according to the invention inhibit the interaction between human CD47 and human SIRP ⁇ .
  • the second binding part of the antibody according to the invention (specifically binding to human CD47) is characterized by a light chain comprising as light chain CDRs a CDRL1 of SEQ ID NO:7, a CDRL2 of SEQ ID NO:8, and a CDRL3 of SEQ ID NO:9 , and a heavy chain comprising as heavy chain CDRs a CDRL1 of SEQ ID NO:1, a CDRL2 of SEQ ID NO:2, and a CDRL3 of SEQ ID NO:3.
  • the second binding part of the antibody according to the invention (specifically binding to human CD47) is characterized by a kappa light chain variable region of SEQ ID NO:10.
  • the second binding part of the antibody according to the invention is characterized by a kappa light chain of SEQ ID NO:11. In one embodiment the second binding part of the antibody according to the invention (specifically binding to human CD47) is characterized by a kappa/lambda light chain of SEQ ID NO:181. In one embodiment the second binding part of the antibody according to the invention (specifically binding to human CD47) is characterized by a heavy chain variable region of SEQ ID NO:4. In one embodiment the second binding part of the antibody according to the invention (specifically binding to human CD47) is characterized by a heavy chain of SEQ ID NO:5. In one embodiment the second binding part of the antibody according to the invention (specifically binding to human CD47) is characterized by a heavy chain of SEQ ID NO:6.
  • binding to CEA refers in the context of the bispecific antibodies according to the invention to specificity for CEACAM5 on the surface of a cell. Binding to CEA (CEACAM5) on cells is preferably measured with gastric adenocarcinoma MKN-45 cells comprising approximately 150.000 CEA copies per cell. The concentration of the antibody according to the invention is varied in an appropriate range in regard to a resulting EC50 value for binding to MKN-45 cells as defined above.
  • membrane-bound human CEA refers to human carcinoembryonic antigen (CEA) that is bound to a membrane-portion of a cell or to the surface of a cell, in particular, the surface of a tumor cell.
  • CEA human carcinoembryonic antigen
  • the term “membrane-bound human CEA” may, in certain circumstances, refer to CEA which is not bound to the membrane of a cell, but which has been constructed so as to preserve the membrane bound CEA epitope to which the antibody according to the invention binds.
  • the terms "cross-reactivity against CEACAM6, specifically binding to CEACAM6, binding to CEACAM6, CEACAM6 binding part” refer in the context of the bispecific antibodies according to the invention that the bispecific antibody according to the invention recognizes specifically CEACAM5 and CEACAM6 on the surface (membrane) of a cell.
  • the bispecific antibodies according to the invention are specifically binding to membrane-bound CEACAM6, when compared to binding to membrane-bound CEA.
  • the ratio of the occupancy of CEACAM5 to CEACAM6 receptors on a cell surface by a given bispecific antibody of the invention is dependent on the binding affinities to CEACAM5 respectively CEACAM6 and can be easily calculated if these binding affinities have been measured, e.g. by SPR.
  • an antibody that specifically binds to CEACAM5 does not bind to carcinoembryonic antigen-related cell adhesion proteins such as, CEACAM1 (UniProtKB - P13688), CEACAM3 (UniProtKB - P40198), CEACAM4 (UniProtKB - 075871), CEACAM7 (UniProtKB - Q14002) and CEACAM8 (CD67 antigen, NCA 95, CD66b, UniProtKB - P31997).
  • the AC CEA antibody according to the invention specifically binds to human CEACAM5 and specifically to cynomolgus CEACAM5.
  • the AC CEA antibody according to the invention specifically binds to human CEACAM5 and cynomolgus CEACAM5 and binds also specifically to human CEACAM6 at similar EC50 as to human CEACAM5 (balanced binding).
  • the bispecific antibody according to the invention specifically binds to human CEACAM5 and specifically to cynomolgus CEACAM5.
  • the bispecific antibody according to the invention specifically binds to human CEACAM5 and cynomolgus CEACAM5 binds also specifically to human CEACAM6 at similar EC50as to human CEACAM5 (balanced binding).
  • the terms "no substantial cross-reactivity against CEACAM1 and/ or CEACAM3, CEACAM4, CEACAM6, CEACAM7 and CEACAM8, non-binding to said CEACAM” refer in the context of the bispecific antibodies according to the invention that such antibodies do not show any relevant binding to said membrane-bound CEACAM at therapeutic plasma concentrations (1 to 1000 nM), when compared to membrane-bound CEACAM5.
  • Non-binding to CEACAM1 and/ or CEACAM3, CEACAM4, CEACAM6 and/or CEACAM8 or to other CEACAM family members can be determined by flow cytometry based measurement of the binding curve to recombinant CHO or PEAK cells expressing said CEACAM or measured by an ELISA based assay measuring the binding to the recombinant CEACAM proteins.
  • the term "not binding to” means in one embodiment that in an ELISA based binding assay (example 8f) to the respective protein a concentration dependent binding curve cannot be established due to very weak or no binding, or the respective binding curve is close to a control.
  • EC50 for binding for such non-binders is 100 or more times higher than EC50 for binding to human CEACAM5.
  • OD values for such non-binders will be usually about equal to that of the limit of detection and/or close to control (human IgG).
  • the terms "does not bind, no binding to" for a compound mentioned herein e.g. human IgG or CEACAM family unrelated tumor-associated proteins, e.g. HER3
  • CEACAM family members are e.g. discussed in Kuespert K Current opinion in Cell Biol 18 (2006) 565-571 .
  • CEA-TCB refers to a bispecific antibody binding to CEA and CD3 as described in US20140242079 (incorporated by reference in its entirety) as SEQ ID NO:1, 2, 21, and 22.
  • SEQ ID NO:106 refers to a bispecific antibody binding to CEA and CD3 as described in US20140242079 (incorporated by reference in its entirety) as SEQ ID NO:1, 2, 21, and 22.
  • amino acid sequences of CEA-TCB are also described as SEQ ID NO:106 to 109 of the present invention.
  • CEA-TCB1 refers to molecule B in the "2+1 IgG CrossFab, inverted" format with charge modifications (VH/VL exchange in CD3 binder, charge modification in CEA binder, humanized CEA binder); SEQ ID NOs 34, 36-38 of WO2017055389 (incorporated by reference in its entirety)).
  • the amino acid sequences of CEA-TCB1 are described as SEQ ID NO:102 to 105 of the present invention.
  • Further CEAxCD3 Mabs are described in WO2007071426 , WO2013012414 , WO2015112534 , WO2017118675 , US20140242079 and WO2017055389 (each of which is incorporated by reference in its entirety).
  • a further CEAxCD3 Mab is RO6958688 (see e.g. Bacac et al Clin. Cancer Res., 22(13), 3286-97 (2016 ).
  • said CEAxCD3 Mab according to the invention is not competitive and/or does not bind to the same epitope of human CEACAM5 as CEA-TCB or CEA-TCB1.
  • CD3 Mab antibody against CD3 refers to human CD3 ⁇ (UniProtKB - P07766 (CD3EHUMAN).
  • the term "antibody against CD3 ⁇ , anti CD3 ⁇ antibody” relates to an antibody specifically binding to CD3 ⁇ .
  • the antibody against CD3 ⁇ is specifically binding to the same epitope as anti-CD3 antibody SP34 (BD Biosciences Catalog No.565983).
  • the antibody against CD3 ⁇ is specifically binding to an epitope of human CD3 ⁇ , comprising the amino acid sequence of SEQ ID NO:118.
  • the antibody against CD3 ⁇ is specifically binding to human CD3 ⁇ and comprises a heavy chain variable region of SEQ ID NO:100 and a light chain variable region of SEQ ID NO:101.
  • the bispecific antibody of the invention does not compete with CEA-TCB and/or CEA-TCB1 for binding on CEA as presented on MKN-45 cells. Therefore CEA-TCB in a concentration of 300 nM (CEA-TCB) or 30 nM (CEA-TCB1) do not shift the EC50 of the phagocytosis index curve of said the bispecific antibody of the invention for MKN-45 cells by more than a factor of 3, in one embodiment towards higher concentrations.
  • 300 nM are a concentration measured in patient plasma at therapeutically effective doses of CEA-TCB (( J.Tabernero et.al., J. Clin. Oncol. 35, 2017 (suppl. Abstr. 3002 )).
  • CEA-TCB1 is in preclinical investigations approx. 10 to 100 times more potent than CEA-TCB (binding affinity, tumor cell lysis, WO2017055389 ), therefore the shift of the EC50 is tested at 30 nM.
  • Non-competition means that EC50 is shifted by less than a factor of 3, in one embodiment to towards higher concentrations, if 300 nM of CEA-TCB are added to the assay. 300 nM are a concentration in the range of therapeutically active doses/plasma-concentrations of CEA x CD3 bispecific antibody (CEA-TCB) ( J.Tabernero et.al., J. Clin. Oncol. 35, 2017 (suppl. Abstr. 3002 )).
  • Non-competition by CEA-TCB1 means that EC50 is shifted by less than a factor of 3 if 30 nM of CEA-TCB 1 are added to the assay.
  • Non-competition means that EC50 is changed by less than a factor of 3 if 300 nM of CEA-TCB are added to the assay. 300 nM are a concentration in the range of therapeutically active doses/plasma-concentrations of CEA x CD3 bispecific antibody (CEA-TCB) ( J.Tabernero et.al., J. Clin. Oncol. 35, 2017 (suppl. Abstr. 3002 )).
  • Non-competition by CEA-TCB1 means that EC50 is changed by less than a factor of 3 if 30 nM of CEA-TCB1 are added to the assay.
  • not competitive means that a second antibody (bispecific antibody against CEAxCD3 ⁇ , like CEA-TCB or CEA-TCB1) in a concentration of 300 nM (CEA-TCB) or 30 nM (CEA-TCB1) does not shift the EC50 of the binding curve of the bispecific antibody of the invention to MKN-45 cells by more than a factor of 3, in one embodiment towards higher concentrations.
  • a second antibody bispecific antibody against CEAxCD3 ⁇ , like CEA-TCB or CEA-TCB1 in a concentration of 300 nM (CEA-TCB) or 30 nM (CEA-TCB1) does not shift the EC50 of the binding curve of the bispecific antibody of the invention to MKN-45 cells by more than a factor of 3, in one embodiment towards higher concentrations.
  • CDR complementarity determining region
  • Kabat numbering refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983 ). Unless otherwise specified, references to the numbering of specific amino acid residue positions in bispecific antibody according to the invention are according to the Kabat numbering system.
  • ADCP antibody-dependent cell-mediated phagocytosis
  • phagocytosis As used herein "phagocytosis, EC50 value of phagocytosis, maximum of phagocytosis, phagocytosis index” according to the invention refer to phagocytosis measured with MKN-45 cells by "imaging".
  • An appropriate imaging method with incubation at an effector (macrophages):target (tumor) cell ratio of e.g. 1:1 or 1:3 and with the "phagocytosis index" as readout (Imaging determined ADCP”) is described in Example 9.
  • phagocytosis of said bispecific antibody means phagocytosis caused/induced by said antibody.
  • Antibody K2AC49 comprises a first binding part, specifically binding to human CEACAM5 and human CEACAM6 and a second binding part, specifically binding to human CD47, whereby the first binding part comprises as heavy chain a heavy chain of SEQ ID NO:5 and as light chain a light chain of SEQ ID NO:85, and that the second binding part comprises as heavy chain a heavy chain of SEQ ID NO:5 and as light chain a light chain of SEQ ID NO:11.
  • antibody K2AC49 comprises as common heavy chain a heavy chain of SEQ ID NO:6.
  • Antibody K2AC50 comprises a first binding part, specifically binding to human CEACAM5 and human CEACAM6 and a second binding part, specifically binding to human CD47, whereby the first binding part comprises as heavy chain a heavy chain of SEQ ID NO:5 and as light chain a light chain of SEQ ID NO:86, and that the second binding part comprises as heavy chain a heavy chain of SEQ ID NO:5 and as light chain a light chain of SEQ ID NO:11.
  • antibody K2AC50 comprises as common heavy chain a heavy chain of SEQ ID NO:6.
  • Antibody K2AC54 comprises a first binding part, specifically binding to human CEACAM5 and a second binding part, specifically binding to human CD47, whereby the first binding part comprises as heavy chain a heavy chain of SEQ ID NO:5 and as light chain a light chain of SEQ ID NO:89, and that the second binding part comprises a heavy chain of SEQ ID NO:5 and as light chain a light chain of SEQ ID NO:11.
  • antibody K2AC54 comprises as common heavy chain a heavy chain of SEQ ID NO:6.
  • Antibody K2AC53 comprises a first binding part, specifically binding to human CEACAM5 and a second binding part, specifically binding to human CD47, whereby the first binding part comprises as heavy chain a heavy chain of SEQ ID NO:5 and as light chain a light chain of SEQ ID NO:88, and that the second binding part comprises as heavy chain a heavy chain of SEQ ID NO:5 and as light chain a light chain of SEQ ID NO:11.
  • Bispecific antibodies K2AC21, K2AC41 to K2AC66 comprise in the second binding part the same heavy chain (SEQ ID NO:5 or 6) and light chain (SEQ ID NO:11), but differ in the first binding part light chain (see table 2, sequence list, Seq ID NO: 185, SEQ ID NO:74 to 95, SEQ ID NO: 167 to 173).
  • phagocytosis can also be measured by a flow cytometry based method as % phagocytosis and at a ratio of e.g. 3 human macrophages to 1 target/tumor-cell ("flow cytometry determined ADCP").
  • human IgG refers to a commercially available clinical-grade homogeneous preparation of human immunoglobulin IgG (e.g. from company Bio-rad.com) that does not bind specifically to CD47 and CEACAM5.
  • Antibodies produced in CHO cells typically have complex biantennary structures with very low or no bisecting-N-acetylglucosamine (bisecting GlcNAc) and high levels of core fucosylation.
  • Bisecting GlcNAc bisecting GlcNAc
  • Overexpression of N-acetylglucosaminyltransferase III has been used to increase the fraction of bisecting GlcNAc that resides on antibodies to improve antibody-dependent cellular cytotoxicity (ADCC).
  • RNAi and gene deletion technologies have also been used to decrease or eliminate the fucose on antibodies to dramatically increase ADCC activity ( Davis J. et al.; Biotechnol. Bioeng. 2001;74:288-294 ; Saba JA, et al.; Anal. Biochem.
  • the bispecific antibody according to the invention is glycoengineered.
  • the glycoengineered bispecific antibody according to the invention has increased ADCC and/or ADCP activity (decreased EC50 and/or higher maximum of phagocytosis index) compared to the bispecific antibody comprising an Fc part included in SEQ ID NO:6 (parent antibody), comprising glycosylation according to a production in a CHO K1 cell line (ATCC® CCL-61TM) at standard conditions (1000ml vessel, temperature 37°C, pH 7.0, impeller speed 80 rpm, minimum dissolved oxygen 30%; cultivation time 14 days).
  • the increase in ADCC is by a factor of 1.2 to 2.0 or even at least 2.0 as compared to said parent.
  • the increase in ADCP is by a factor of at least 3 or even 5 or more as compared to said parent.
  • polypeptide having GnTIII activity refers to polypeptides that are able to catalyze the addition of a N-acetylglucosamine (GlcNAc) residue in ⁇ -1-4 linkage to the ⁇ -linked mannoside of the trimannosyl core of N-linked oligosaccharides, eg. ⁇ -1,4-mannosyl-glycoprotein4- ⁇ -N-acetylglucosaminyl-transferase (EC 2.4.1.144).
  • GlcNAc N-acetylglucosamine
  • FUT8 refers to ⁇ 1,6-fucosyltransferase (EC:2.4.1.68).
  • effector function Fc-mediated cellular cytotoxicity
  • Fc region a native sequence Fc region or amino acid sequence variant Fc region
  • antibody effector functions include, but are not limited to, Fc receptor binding affinity, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune-complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors, etc.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • cytokine secretion immune-complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors, etc.
  • immune mechanism is leading to the lysis of "targeted cells” by "human immune effector cells.”
  • GDP-6-deoxy-D-lyxo-4-hexulose reductase refers to an enzyme (UniProtKB - F0J3S3) which uses GDP-6-deoxy-D-lyxo4-hexulose as a substrate.
  • GDP-6-deoxy-D-lyxo-4-hexulose reductase reduces the substrate GDP-6-deoxy-D-lyxo-4-hexulose to GDP-D-rhamnose.
  • GDP-D-Rhamnose is a nucleotide sugar donor for D-rhamnosylation in bacteria and does not occur in vertebrates.
  • Vertebrate cells also lack specific rhamnosyltransferases so that GDP-D-Rhamnose cannot be incorporated into nascent glycostructures of glycoproteins or glycolipids within vertebrate cells (see US8642292 , incorporated herein by reference).
  • glycoengineered antibody refers to a bispecific antibody according to the invention which comprises a reduced amount of fucosylated and/or bisecting oligosaccharides attached to the Fc region of said antibody, usually at amino acid Asn297, compared to a parent antibody.
  • inhibiting fucose synthesis refers to a method blocking the de novo synthesis of fucose in the host cell by use of RMD and - in one embodiment - culturing in addition the host cell in fucose free medium as described in US8642292 .
  • parent antibody, parent bispecific antibody in the context of glycoengineering refers to a bispecific antibody according to the invention which comprises the same amino acid composition as the glycoengineered antibody but is non-glycoengineered.
  • parent antibody and the glycoengineered antibody are produced in the same host cell, but in the first case in the host cell without glycoengineering, and in the second case in the same host cell but engineered
  • human immune effector cells refers to a population of leukocytes that display Fc receptors on their surfaces, through which they bind to the Fc-region of antigen binding molecules or of Fc-fusion proteins and perform effector functions.
  • a population may include, but is not limited to, peripheral blood mononuclear cells (PBMC) and/or natural killer (NK) cells and/or macrophages.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer
  • the term "increased Fc-mediated cellular cytotoxicity” is defined as either an increase in the number of "targeted cells” that are lysed in a given time, at a given concentration of the bispecific antibody of the invention in the medium surrounding the target cells, by the mechanism of Fc-mediated cellular cytotoxicity defined above, and/or a reduction in the concentration of the bispecific antibody of the invention, in the medium surrounding the target cells, required to achieve the lysis of a given number of "targeted cells” in a given time, by the mechanism of Fc-mediated cellular cytotoxicity.
  • the increase in Fc-mediated cellular cytotoxicity is relative to the cellular cytotoxicity mediated by the same bispecific antibody of the invention produced by the same type of host cells, using the same standard conditions, but that has not been produced by host cells engineered to have an altered pattern of glycosylation (e.g., by inhibiting fucose synthesis, by expressing glycosyltransferase, GnTIII, or other glycosyltransferases or FUT8 disruption) by the methods described herein.
  • an altered pattern of glycosylation e.g., by inhibiting fucose synthesis, by expressing glycosyltransferase, GnTIII, or other glycosyltransferases or FUT8 disruption
  • expression vector refers to one or more vectors which comprise the heavy and light chains of the antibody according to the invention in an appropriate manner as known from the state of the art.
  • host cells engineered by targeted disruption of the FUT8 gene refers to host cells capable of expressing an antibody according to the invention and being in addition glycoengineered by targeted disruption of the FUT8 gene as described e.g. in US8067232 , US7425446 , US6946292 (each of which is incorporated by reference in its entirety), and Yamane-Ohnuki N. et al., Biotech. Bioeng.; 87 (2004) 614-622 .
  • An antibody according to the invention expressed in such host cell comprises a Fc region comprising complex N-glycoside-linked sugar chains bound to the Fc region, which comprise a reducing end which contains an N-acetylglucosamine, wherein the sugar chains do not contain fucose bound to the 6 position of N-acetylglucosamine in the reducing end of the sugar chains.
  • the present invention is further directed to a method for the production of a bispecific antibody according to the present invention characterized in comprising nonfucosylation of 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%, that are produced by a host cell, comprising expressing in said host cell a nucleic acid encoding a bispecific antibody of the invention and a nucleic acid encoding a polypeptide with a glycosyltransferase activity, or a vector comprising such nucleic acids.
  • Genes with glycosyltransferase activity include (1,4)-N-acetylglucosaminyltransferase III (GnTIII), ⁇ -mannosidase II (Manll), (1,4)-galactosyltransferase (GalT), (1,2)-N-acetylglucosaminyltransferase I (GnTI), and ⁇ (1,2)-N-acetylglucosaminyltransferase II (GnTII).
  • a combination of genes with glycosyltransferase activity is expressed in the host cell (e.g., GnTIII and Man II).
  • the method also encompasses expression of one or more polynucleotide(s) encoding the bispecific antibody in a host cell in which a glycosyltransferase gene has been disrupted or otherwise deactivated (e.g., a host cell in which the activity of the gene encoding al-6 core fucosyltransferase has been knocked out).
  • the bispecific antibodies of the present invention can be produced in a host cell that further expresses a polynucleotide encoding a polypeptide having GnTIII activity to modify the glycosylation pattern.
  • the polypeptide having GnTIII activity is a fusion polypeptide comprising the Golgi localization domain of a Golgi resident polypeptide.
  • the expression of the bispecific antibodies of the present invention in a host cell that expresses a polynucleotide encoding a polypeptide having GnTIII activity results in bispecific antibodies with increased Fc receptor binding affinity and increased effector function.
  • the present invention is further directed to a method for the production of a bispecific antibody according to the present invention characterized in comprising non-fucosylation of 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%, that are produced by a host cell, comprising expressing in said host cell a nucleic acid encoding a bispecific antibody of the invention and a disrupted FUT8 gene.
  • the present invention is further directed to a method for the production of a bispecific antibody according to the present invention characterized in comprising non-fucosylation of 80% to 100%, or 90% to 100%, that are produced by a host cell, comprising expressing in said host cell a nucleic acid encoding a bispecific antibody of the invention by inhibiting fucose synthesis in said host cell.
  • the bispecific antibodies with altered glycosylation produced by the host cells of the invention exhibit increased Fc receptor binding affinity and/or increased effector function as a result of the modification of the host cell (e.g., by inhibiting fucose synthesis in said host cell or by expression of a glycosyltransferase gene).
  • the increased Fc receptor binding affinity is increased binding to a Fc ⁇ activating receptor, such as the Fc ⁇ RIIIa receptor.
  • the percentage of nonfucosylated oligosaccharides is 50% to 100%, specifically 60% to 100%, 70% to 100%, and more specifically, 80% to 100%.
  • the nonfucosylated oligosaccharides may be of the hybrid or complex type.
  • the bispecific antibody produced by the methods of the invention has an increased proportion of bisected oligosaccharides in the Fc region as a result of the modification of its oligosaccharides by the methods of the present invention.
  • the percentage of bisected oligosaccharides is 50% to 100%, specifically 50%, 60% to 70%, and more specifically, 80%.
  • the bispecific antibody produced by the host cells and methods of the invention has an increased proportion of bisected, nonfucosylated oligosaccharides in the Fc region.
  • the bisected, nonfucosylated oligosaccharides may be either hybrid or complex.
  • the term "host cell” covers any kind of cellular system which can be engineered to generate the bispecific antibodies of the present invention.
  • the host cell is engineered to allow the production of an antigen binding molecule with modified glycoforms.
  • the host cells have been further manipulated to express increased levels of one or more polypeptides having GnTIII activity.
  • Host cells include cultured cells, e.g., mammalian cultured cells, such as CHO cells (see above), BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
  • Host cells for the production of glycoengineered bispecific antibodies of the present invention have been described e.g. in US6602684 , US20040241817 , US20030175884 ; and WO 2004065540 .
  • the bispecific antibodies of the present invention can alternatively be glycoengineered to have reduced fucose residues in the Fc region according to the techniques disclosed in US2003/0157108 , EP1176195 , WO2003084570 , WO2003085119 and US2003/0115614 , US2004/093621 , US2004/110282 , US2004/110704 , US2004/132140 (each of which is incorporated by reference in its entirety).
  • Glycoengineered bispecific antibodies of the invention may also be produced in expression systems that produce modified glycoproteins, such as those described in WO2003/056914 , WO2004/057002 , and WO2004/024927 (each of which is incorporated by reference in its entirety).
  • the antibody according to the invention comprises one or two or three amino acid substitutions in the Fc region ("Fc amino acid substitution") selected from the group consisting of mono-substitutions S239D, I332E, G236A, of bi-substitutions I332E and G236A, S239D and I332E ("DE substitution"), S239D and G236A, and triple-substitution S329D and I332E and G236A ("DEA substitution”); ( Richards JO, et al., Mol. Cancer Ther. 7 (2008) 2517-2527 ). Due to different counting of a heavy chain, these amino acid numbers can be different for +/one, two or three amino acids, but with the same shift for all three.
  • Fc amino acid substitution selected from the group consisting of mono-substitutions S239D, I332E, G236A, of bi-substitutions I332E and G236A, S239D and I332E (“DE substitution"), S239D and G236
  • parent antibody, parent bispecific antibody in the context of Fc substitution refers to a bispecific antibody according to the invention which comprises the same amino acid composition as the Fc substituted antibody, but without said substitution(s).
  • parent antibody and the Fc substituted antibody are produced - as in the case of glycoengineered antibodies - in the same host cell under the same conditions, but in the first case in the host cell without Fc substitution, and in the second case in the same host cell but with such Fc substitution(s).
  • a useful host cell line is e.g. CHO-K1.
  • parent antibody in the context of a bispecific antibody according to the invention which comprises Fc substitution and is glycoengineered, such parent antibody therefore is the respective bispecific antibody which comprises the same amino acid composition as the Fc substituted antibody, but without said substitution(s) and is not glycoengineered.
  • ADCC and/or ADCP activity of the bispecific antibody is increased by amino acid substitution of the Fc part in combination with glycoengineering of the Fc part compared ADCC and/or ADCP activity of the respective parent antibody.
  • the invention comprises therefore in one embodiment a bispecific antibody specifically binding to human CEACAM5 and human CD47, characterized in comprising one or two or three amino acid substitutions in the Fc region ("Fc amino acid substitution") selected from the group consisting of mono-substitutions S239D, I332E, G236A, of bi-substitutions I332E and G236A, S239D and I332E, of triple-substitutions S329D and I332E and G236A and comprising non-fucosylation of the Fc part of 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%.
  • Fc amino acid substitution selected from the group consisting of mono-substitutions S239D, I332E, G236A, of bi-substitutions I332E and G236A, S239D and I332E, of triple-substitutions S329D and I332E and G236A and comprising non-fucosylation of the Fc
  • Example 9 describes assays used for the determination of ADCC activity and also of ADCP activity ADCC can be measured by an in vitro ADCC assay as follows:
  • ADCC is defined as either an increase in the maximum percentage of specific lysis observed within the bispecific antibody concentration range tested above, and/or a reduction in the concentration of bispecific antibody required to achieve one half of the maximum percentage of specific lysis (EC50) observed within the bispecific antibody concentration range tested above.
  • the increase in ADCC is relative to the ADCC, measured with the above assay, mediated by the same bispecific antibody, produced by the same type of host cells, using the same standard production, purification, formulation and storage methods, but that has not been produced by host cells engineered a) to inhibit fucose synthesis in said host cell, b) to overexpress GnTIII or c) by host cells engineered by targeted disruption of the FUT8 gene ("parent antibody”).
  • the increase in ADCC is relative to the ADCC measured with the parent bispecific antibody not carrying the substitution(s).
  • the increase in ADCC is relative to the ADCC measured with the parent non glycoengineered, bispecific antibody not carrying the substitution(s).
  • the CEACAM x CD47 bispecific antibodies according to the invention are optimized for treatment of solid tumors mainly by macrophages mediated phagocytosis of the tumor cells, either in monotherapy or in combination therapy especially together with a CEAxCD3 T-cell bispecific antibody like CEA-TCB or CEA-TCB1 and/or PD-1 axis antagonist.
  • the antibody according to the invention and the CEAxCD3 T-cell bispecific antibody can be administered as described below.
  • the disease resp. solid tumor is a cancer that expresses or even overexpresses CEACAM5 or CEACAM5 and CEACAM6, including but not limited to the group of colorectal tumors, non-small cell lung tumors, gastric tumors, pancreatic tumors and breast tumors.
  • the tumor is a colorectal tumor.
  • the tumor is a gastric tumor.
  • the tumor is a gastric tumor expressing CEACAM5 or CEACAM5 and CEACAM6 and HER-2. All therapeutic applications methods of use, uses, combinations, etc. described herein are especially embodiments for the treatment of these tumors/diseases.
  • the inventors recognize that the antibodies according to the invention show low or no ADA formation potential respectively loss of exposure due to neutralizing ADA respectively loss of efficacy.
  • the invention provides a method of treating carcinomas (cancer, tumors, for example, human carcinomas), especially CEACAM5 or CEACAM5 and CEACAM6 expressing tumors, in vivo.
  • This method comprises administering to a subject a pharmaceutically effective amount of a composition containing a bispecific antibody of the invention.
  • subject is meant a human subject, in one embodiment a patient suffering from cancer/tumor/carcinoma.
  • CEACAM5 or CEACAM5 and CEACAM6 expression in various tumor entities is generally very high, especially in colorectal carcinoma, pancreatic adenocarcinoma, gastric cancer, non-small cell lung cancer, breast cancer, head and neck carcinoma, uterine and bladder cancers among others.
  • CEACAM5 or CEACAM5 and CEACAM6 is mainly expressed in a polarized pattern on the apical surface of the cells. This polarized expression pattern limits the accessibility by anti-CEA mono or bispecific antibodies which are administered systemically and therefore potential toxicity. Together with the low affinity CD47 binding of the antibody of the invention this leads to no or limited phagocytosis of such normal cells by the antibody of the invention.
  • CEACAM5 or CEACAM5 and CEACAM6 is expressed equally over the whole cell surface of the cancer cells that means cancer cells are much better accessible to an antibody of the invention than normal, healthy cells and can be selectively killed by the CEAxCD47 bispecific antibodies of the invention respectively by the combinations mentioned above.
  • the bispecific antibodies of this invention can be used in monotherapy for the treatment of advanced solid tumors, in one embodiment CEACAM5 or CEACAM5 and CEACAM6 expressing tumors.
  • a bispecific antibody according to the invention is used in combination with a CEAxCD3 Mab in simultaneous, separate, or sequential combination.
  • a bispecific antibody according to the invention is used in combination with a CEAxCD3 Mab and/or a PD-1 axis antagonist in simultaneous, separate, or sequential combination.
  • a bispecific antibody according to the invention is used in combination with a PD-1 axis antagonist in simultaneous, separate, or sequential combination.
  • Such PD-1 axis antagonists are described e.g. in WO2017118675 .
  • CEAxCD3 Mabs are in clinical development (CEA-TCB and CEA-TCB1; see clinicaltrials.gov; RO6958688 in NCT3866239 and RO7172508 in NCT03539484).
  • MEDI-565 was in clinical development but no active clinical trial could be identified in clinicaltrials.gov.
  • antibody CEA-TCB or CEA-TCB 1 is used as bispecific antibody against CEA and CD3, antibody CEA-TCB or CEA-TCB 1 is used.
  • CEA-TCB has been derived from anti-CEA antibody PR1A3 (see e.g. EP2681244B1 ). This antibody binds to the so called B3 domain of CEA.
  • CEA-TCB has a low nM binding affinity to CEA and shows efficacy in high doses (between 40 and 600 mg per dose and patient; (see e.g. J.Tabernero et.al., J. Clin. Oncol. 35, 2017 (suppl. Abstr. 3002 )). At highest doses nearly all CEA targets on the cell surfaces are occupied by the CEA-TCB.
  • Combination of CEA-TCB or CEA-TCB 1 and CEAxCD47 generates therapeutic plasma levels of both drugs at the same time and achieves best results (additive or even synergistic), if both drugs are non-competitive for the CEA antigen.
  • the terms "combination, simultaneous, separate, or sequential combination" of a an antibody according to the invention and a second bispecific antibody, binding to human CEA and human CD3 ⁇ refer to any administration of the two antibodies (or three antibodies in case of the combination of an antibody of the invention, a CEAxCD3 Mab and a PD-1 axis antagonist), either separately or together, where the two or three antibodies are administered as part of an appropriate dose regimen designed to obtain the benefit of the combination therapy, for example in separate, sequential, simultaneous, concurrent, chronologically staggered or alternating administration.
  • the two or three antibodies can be administered either as part of the same pharmaceutical composition or in separate pharmaceutical compositions.
  • the antibody according to the invention can be administered prior to, at the same time as, or subsequent to the administration of the second bispecific antibody, or in some combination thereof.
  • the second bispecific antibody can be administered prior to, at the same time as, or subsequent to, each administration of the antibody of the invention or some combination thereof, or at different intervals in relation to the treatment with the antibody of the invention, or in a single dose prior to, at any time during, or subsequent to the course of treatment with the antibody of the invention.
  • the antibody according to the invention and the second bispecific antibody are administered in alternating administration, in one embodiment in intervals of 6 to 15 days between administration of the antibody of the invention and the second antibody. In such alternating administration the first dose can be the antibody of the invention or the second antibody.
  • PD-1 axis antagonist refers to an anti-PD-1 antibody or an anti-PD-Ll antibody.
  • Anti-PD-1 antibodies are e.g. pembrolizumab (Keytruda®, MK-3475), nivolumab, pidilizumab, lambrolizumab, MEDI-0680, PDR001, and REGN2810.
  • Anti-PD-1 antibodies are described e.g.
  • Anti-PD-Ll antibodies are e.g. atezolizumab, MDX-1 105, durvalumab and avelumab. Anti-PD-Ll antibodies are e.g. described in WO2015026634 , WO2013/019906 , WO2010077634 , US8383796 , WO2010077634 , WO2007005874 , and WO2016007235 (each of which is incorporated by reference in its entirety).
  • both compounds may be present in one single dosage form or in separate dosage forms, for example in two different or identical dosage forms.
  • both antibodies if desired by the physician, can be administered simultaneously. If the antibody of the invention and the second antibody are competing in regard to CEACAM5, in one embodiment both antibodies are administered in alternating administration.
  • the antibody of the invention will typically be administered to the patient in a dose regimen that provides for the most effective treatment of the cancer (from both efficacy and safety perspectives) for which the patient is being treated, as known in the art.
  • tumor cells are attacked at the same time by T-cells and macrophages, to achieve full therapeutic potential of this approach, CEA-CD3 and CEAxCD47 bispecific antibody have to be non-competitive regarding binding to CEA on cell surface.
  • the amount of the antibody administered and the timing of the administration of the antibody of the invention can depend on the type (e.g. gender, age, weight) and condition of the patient being treated, the severity of the disease or condition being treated, and on the route of administration.
  • the antibody of the invention and the second antibody can be administered to a patient in doses ranging from 0.1 to 100 mg/kg of body weight per day or per week in single or divided doses, or by continuous infusion.
  • each of the antibodies of the invention and the second antibody is administered to a patient in doses ranging from 0.1 to 20 mg/kg.
  • dosage levels below the lower limit of the aforesaid range may be adequate, while in other cases still larger doses may be employed without causing any harmful side effect.
  • half-life of the antibody refers to the half-life of said antibody as measured in a usual pharmacokinetic assay, e.g. as described in example 17.
  • An antibody according to the invention and the second bispecific antibody against CEA and CD3 have elimination half-life of 3-14 days.
  • the invention is also directed to use of the bispecific antibody according to the invention in the treatment of disease, particularly cell proliferation disorders wherein CEACAM5 or CEACAM5 and CEACAM6 is expressed, particularly wherein CEACAM5 or CEACAM5 and CEACAM6 is abnormally expressed (e.g., overexpressed or expressed in a different pattern on the cell surface) compared to normal tissue of the same cell type.
  • diseases include, but are not limited to colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer and breast cancer.
  • CEACAM5 or CEACAM5 and CEACAM6 expression levels may be determined by methods known in the art (e.g., via immunohistochemistry assay, immunofluorescence assay, immunoenzyme assay, ELISA, flow cytometry, radioimmunoassay etc.).
  • bispecific antibodies of the present invention can be used for targeting cells in vivo or in vitro that expresses CEACAM5 or CEACAM5 and CEACAM6.
  • the bispecific antibodies of the invention are particularly useful in the prevention of tumor formation, eradication of tumors and inhibition of tumor growth or metastasis via the induction of ADCP and ADCC of tumor cells.
  • the bispecific antibodies of the invention can be used to treat any tumor expressing CEACAM5 or CEACAM5 and CEACAM6 A.
  • Particular malignancies that can be treated with the bispecific antibodies of the invention include, but are not limited to, colorectal cancer, non- small cell lung cancer, gastric cancer, pancreatic cancer and breast cancer.
  • the bispecific antibodies of the invention are administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form such as those discussed below, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the bispecific antibodies of the invention also are suitably administered by intra tumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects.
  • bispecific antibodies of the invention For the treatment of disease, the appropriate dosage of bispecific antibodies of the invention will depend on the type of disease to be treated, the severity and course of the disease, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the bispecific antibody of the invention is suitably administered to the patient at one time or over a series of treatments.
  • the present invention provides a method for selectively killing tumor cells expressing CEACAM5 or CEACAM5 and CEACAM6.
  • This method comprises interaction of the bispecific antibodies of the invention with said tumor cells.
  • These tumor cells may be from a human carcinoma including colorectal carcinoma, non-small cell lung carcinoma (NSCLC), gastric carcinoma, pancreatic carcinoma and breast carcinoma.
  • NSCLC non-small cell lung carcinoma
  • gastric carcinoma pancreatic carcinoma
  • breast carcinoma breast carcinoma
  • the invention is directed to the use of the bispecific antibodies of the invention for the manufacture of a medicament for treating a disease related to abnormal CEACAM5 or CEACAM5 and CEACAM6 expression.
  • the disease is a cancer that expresses or even overexpresses CEACAM5 or CEACAM5 and CEACAM6, including but not limited to colorectal tumor, non-small cell lung tumor, gastric tumor, pancreatic tumor and breast tumor.
  • the tumor is a colorectal tumor.
  • compositions Compositions, Formulations, Dosages, and Routes of Administration
  • the present invention is directed to pharmaceutical compositions comprising the bispecific antibodies of the present invention and a pharmaceutically acceptable carrier.
  • the present invention is further directed to the use of such pharmaceutical compositions in the method of treatment of disease, such as cancer, or in the manufacture of a medicament for the treatment of disease, such as cancer.
  • the present invention is directed to a method for the treatment of disease, and more particularly, for the treatment of cancer, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the invention.
  • the present invention encompasses pharmaceutical compositions, combinations and methods for treating human carcinomas, tumors, as defined above.
  • the invention includes pharmaceutical compositions for use in the treatment of human carcinomas comprising a pharmaceutically effective amount of an antibody of the present invention and a pharmaceutically acceptable carrier.
  • the bispecific antibody compositions of the invention can be administered using conventional modes of administration including, but not limited to, intravenous, intraperitoneal, oral, intralymphatic or direct intratumoral administration. Intravenous administration or subcutaneous administration are preferred.
  • therapeutic formulations containing the bispecific antibodies of the invention are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers ( Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980 )), in the form of lyophilized formulations or liquid formulations.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • the most effective mode of administration and dosage regimen for the pharmaceutical compositions of this invention depends upon the severity and course of the disease, the patient's condition and response to treatment and the judgment of the treating physician. Accordingly, the dosages of the compositions may be flat doses or may be adapted to the individual patient, e.g. the body weight. Nevertheless, an effective dose of the compositions of this invention will generally be in a range from 0.1 to 20 mg/kg.
  • the bispecific antibodies of this invention have a molecular weight in a magnitude of 150kD per Mol. They carry in one embodiment a Fc part.
  • the elimination half-life in patients is in a range of 3 to 14 days. This half-life allows for, but not limited to administration once a day, once a week, or once every two weeks.
  • bispecific antibodies of the present invention and their respective compositions may be in a variety of dosage forms which include, but are not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microvesicles, liposomes, and injectable or infusible solutions.
  • dosage forms include, but are not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microvesicles, liposomes, and injectable or infusible solutions.
  • the preferred form depends upon the mode of administration and the therapeutic application.
  • composition comprising a bispecific antibody of the present invention will be formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context include the particular disease or disorder being treated, the particular mammal being treated, the clinic condition of the individual patient, the cause of the disease or disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is a bispecific antibody of the invention.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a bispecific antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such
  • the sequence corresponding to the extracellular domain of human CD47 is amplified from human cDNA by polymerase chain reaction (PCR) using specific oligonucleotides.
  • the amplification product is gel-purified and cloned into the pEAK8 mammalian expression vector (Edge Biosystems, Gaithersburg, Md.).
  • the vector is further modified to introduce an AvitagTM (Avidity, Denver Colo.) and a hexa-histidine tag at the C-terminus allowing for single site biotinylation of the protein and purification by IMAC (Immobilized Metal Ion Affinity Chromatography), respectively.
  • the constructs are verified by DNA sequencing.
  • the plasmid is then transfected into mammalian cells using a liposome-based transfection reagent such as Lipofectamine2000 (Thermofisher Scientific).
  • a liposome-based transfection reagent such as Lipofectamine2000 (Thermofisher Scientific).
  • the transfection step requires only small quantities of DNA and cells, typically 2x10 5 cells and 2 ⁇ g of plasmid DNA per well and the transfection carried out in a 6-well plate.
  • different mammalian cell lines can be used, in the examples given below, transformed human embryo kidney monolayer epithelial cells (PEAK cells) are transfected.
  • PEAK cells transformed human embryo kidney monolayer epithelial cells
  • These cells stably express the EBNA-1 gene, further supporting the episomal replication process, are semi-adherent and can be grown under standard cell culture conditions (5% CO 2 ; 37°C in DMEM medium supplemented with 10% fetal calf serum). After 24 h, cells are placed under selective conditions by adding medium containing 0.5-2 ⁇ g/mL puromycin: cells harboring the episomal vector are resistant to this antibiotic.
  • the CELLineTM is a two-compartment bioreactor that can be used in a standard cell culture incubator.
  • the smaller compartment (15 ml) contains the cells and is separated from a larger (one liter) medium containing compartment by a semi-permeable membrane with a cut-off size of 10 kDa (Bruce et al. 2002, McDonald et al. 2005).
  • This system allows for the diffusion of nutrients, gazes and metabolic waste products, while retaining cells and secreted proteins in the smaller compartment.
  • the culture is maintained for 7-10 days before harvest of the supernatant. As the medium contains serum, the cells maintain good viability and several production runs can be generated using the same cells and containers.
  • the cell culture supernatants are clarified by centrifugation.
  • the supernatant is then supplemented with 100 mM imidazole and loaded on Ni-NTA affinity chromatography resin (Qiagen).
  • Ni-NTA affinity chromatography resin Qiagen
  • the relatively high concentration of imidazole minimizes binding of contaminants to the resin.
  • proteins are eluted at a flow rate of 2 mL/min using a 30 mL imidazole gradient (20-400 mM imidazole) on an AKTA Prime chromatography system (Amersham Pharmacia Biotech).
  • the elution gradient further improves the purity of the recombinant protein but can be replaced by a step elution approach if a chromatography system is not available.
  • the eluted fractions can be analyzed by SDS-PAGE or ELISA to determine their content in recombinant protein.
  • the fractions of interest are pooled and desalted on Amicon 10KD columns (Millipore) equilibrated with phosphate buffered saline or another appropriate buffer.
  • the desalted proteins can then be quantified using various techniques and their purity analyzed by SDS-PAGE.
  • Recombinant CD47 is biotinylated in vitro using biotin ligase (Avidity, Denver Colo.) according to manufacturer's instructions. After desalting the biotinylation level is evaluated by pull-down assays using streptavidin magnetic beads and SDS-PAGE analysis.
  • CEACAM5 complete extracellular domain
  • A3-B3 domains of CEACAM5 were synthesized by Eurofins and Twist Bioscience. These synthetic genes were subcloned into the pEAK8 mammalian expression vector (Edge Biosystems, Gaithersburg, Md.). The vectors were modified to introduce an AvitagTM (Avidity, Denver Colo.) and either a hexa-histidine tag, a human FC region or a mouse FC region at the C-terminus. Constructs were verified by DNA sequencing.
  • AvitagTM Asvidity, Denver Colo.
  • Vectors encoding for the full-length version of human CEACAM 1, 3, 4, 5, 6, 7, 8, 18, 19, 20, 21 and cynomolgus CEACAM5 were also generated for expression at the cell surface of PEAK and/or CHO cells.
  • the soluble, full-length human CEACAM16 was also similarly cloned.
  • scFv phage libraries (10 12 Pfu) are blocked with PBS containing 3% (w/v) skimmed milk for one hour at room temperature on a rotary mixer.
  • Blocked phage is deselected on streptavidin magnetic beads (DynabeadsTM M-280) for one hour at room temperature on a rotary mixer.
  • Deselected phage is incubated with 100 nM of either biotinylated human CEACAM5 or the A3-B3 domain captured on streptavidin magnetic beads for two hours at room temperature on a rotary mixer. Beads are captured using a magnetic stand followed by five washes with PBS/0.1% Tween 20 and two washes with PBS.
  • Phage is eluted with 100 nM TEA for 30 minutes at room temperature on a rotary mixer. Eluted phage and beads are neutralized with Tris-HCl 1M pH 7.4 and directly added to 10 ml of exponentially growing TG1 cells and incubated for one hour at 37°C with slow shaking (90 rpm). An aliquot of the infected TG1 is serial diluted to titer the selection output. The remaining infected TG1 are spun at 3800 rpm for 10 minutes and resuspended in 2 ml 2xTY and spread on 2xTYAG (2xTY medium containing 100 ⁇ g/ml ampicillin and 2% glucose) agar Bioassay plates.
  • 50 ⁇ l of cell suspension obtained from previous selection rounds are added to 50 ml of 2xTYAG and grown at 37°C with agitation (240 rpm) until an OD 600 of 0.3 to 0.5 is reached.
  • the culture is then super-infected with 1.2x10 11 M13K07 helper phage and incubated for one hour at 37°C (90 rpm).
  • the medium is changed by centrifuging the cells at 3800 rpm for 10 minutes, removing the medium and resuspending the pellet in 50 ml of 2xTYAK (2xTY medium containing 100 ⁇ g/ml ampicillin; 50 ⁇ g/ml kanamycin).
  • the culture is then grown overnight at 30°C (240 rpm). The next day, the phage containing supernatant is used for the next round of selection.
  • Phage containing supernatants are blocked with PBS containing 3% (w/v) skimmed milk for one hour at room temperature on a rotary mixer. Blocked phage is then deselected for one hour on MKN45 CEACAM5 KO that do not express human CEACAM5. Deselected phage is incubated with 2x10 7 MKN45 cells expressing CEACAM5 (blocked in PBS 3% BSA 0.1% NaN 3 ) for two hours at room temperature with gentle shaking. Cells are pelleted and washed six times with PBS. Bound phage is eluted with 76 mM citric acid and shaking for 10 minutes.
  • Individual clones are inoculated into a deep-well microtiter plate containing 0.9 ml per well of 2xTYAG medium (2xTY medium containing 100 ⁇ g/ml ampicillin, 0.1% glucose) and grown at 37°C for 5-6 hours (240 rpm). 100 ⁇ l per well of 0.2 mM IPTG in 2xTY medium are then added to give a final concentration of 0.02 mM IPTG. The plate is incubated overnight at 30°C with shaking at 240 rpm. The deep-well plate is centrifuged at 3200 rpm for 10 minutes at 4°C and the supernatant carefully removed.
  • 2xTYAG medium 2xTY medium containing 100 ⁇ g/ml ampicillin, 0.1% glucose
  • the pellets are resuspended in 150 ⁇ l TES buffer (50 mM Tris-HCl (pH 8), 1 mM EDTA (pH 8), 20% sucrose, complemented with Complete protease inhibitor, Roche).
  • a hypotonic shock is produced by adding 150 ⁇ l of diluted TES buffer (1:5 TES:water dilution) and incubation on ice for 30 minutes. The plate is centrifuged at 4000 rpm for 10 minutes at 4°C to pellet cells and debris. The supernatants are carefully transferred into another microtiter plate and kept on ice for immediate testing in functional assays or binding assays.
  • scFv for binding to CEACAM5 is tested in a homogenous assay using CellInsightTM technology.
  • the following reagents are mixed in each well of a 384 clear bottom well plate (Corning): 30 ⁇ l of a streptavidin polystyrene bead suspension (Polysciences; 3000 beads/well) coated with either biotinylated CEACAM5, biotinylated domain A3-B3 or biotinylated NusA for a control protein; 60 ⁇ l of blocked scFv periplasmic preparation; 10 ⁇ l of detection buffer (PBS containing mouse anti-c-myc antibody at 5 ⁇ g/ml; anti-mouse Fc AlexaFluor® 647 diluted 1:200).
  • detection buffer PBS containing mouse anti-c-myc antibody at 5 ⁇ g/ml
  • the 384-well plate After mixing at 600 rpm for 5 minutes, the 384-well plate is incubated at room temperature and read after 2 hours on a CellInsightTM CX5 High-Content Screening platform (ThermoFisher Scientific). Clones expressing scFv giving a specific signal for CEACAM5 and not NusA are selected for further analysis or sequencing.
  • CEACAM1, CEACAM6 and other CEACAMs Binding to CEACAM1, CEACAM6 and other CEACAMs can be measured in the same manner.
  • Single clones are inoculated into a 96-deep-well microtiter plate containing 1 ml LBAG medium (LB medium with 100 ⁇ g/ml ampicillin and 2% glucose) per well and grown overnight at 37°C, 240 rpm. DNA is extracted using the Zyppy-96 Plamis Miniprep kit (Zymo Research) and sequenced.
  • LBAG medium LB medium with 100 ⁇ g/ml ampicillin and 2% glucose
  • scFv candidates with the desired binding properties are reformatted into IgG1 antibodies and expressed by transient transfection into PEAK cells.
  • the VH and VL sequences of selected scFv are amplified with specific oligonucleotides and cloned into an expression vector containing the heavy and light chain constant regions and the constructions are verified by sequencing.
  • the expression vectors are transfected into mammalian cells using Lipofectamine 2000 (Thermofisher Scientific) according to manufacturer's instructions. Briefly, 3.5x10 6 PEAK cells are cultured in T75 flasks in 25 ml culture media containing fetal bovine serum.
  • Transfected cells are cultured for 5-6 days at 37°C, IgG1 production is quantified by OctetRED96 instrument.
  • the supernatant is harvested for IgG1 purification on FcXL affinity resin (Thermofisher Scientific) according to manufacturer's instructions. Briefly, supernatants from transfected cells are incubated overnight at 4°C with an appropriate amount of FcXL resin. After resin wash by PBS, samples are loaded on Amicon Pro column and the IgG consequently eluted in 50 mM Glycine pH3.5. The eluted IgG fraction is then dialyzed by Amicon 50kDa against Histidine NaCl pH6.0 buffer and the IgG1 content is quantified by absorption at 280 nm. Purity and IgG1 integrity are verified by Agilent Bioanalyzer manufacturer (Agilent Technologies, Santa Clara, Calif., USA).
  • Mutations are introduced in some positions in the CDR1, CDR2 and/or in the CDR3 of the variable light chain (VL) of the IgG1 candidate (parenteral VL) to be optimized, thereby creating lead optimization phage libraries.
  • VL variable light chain
  • variants with improved binding affinities are selected.
  • the diversity in the CDRs is introduced in the parental VL sequence by using synthetic degenerated oligonucleotides and PCR assembling to generate VL fragments that are cloned into the expression vector pNDS containing the fixed variable heavy chain (VH).
  • the plasmids thus created are transformed into TG1 bacteria by electroporation. Lead optimization libraries have typically diversities of ⁇ 108-109 variants.
  • the transformed TG1 are then rescued by the helper phage M13KO7 to produce phage displaying scFv at the surface. These phage are used for rounds of phage display selections (for Screening/Srios see Example 4) where the stringency was maintained relatively high using target concentrations in the 1-10 nM range in order to enable enrichment for candidates with higher affinities.
  • CEACAM5 monoclonal antibodies can be shown by flow cytometry using PEAK and/or CHO cells transfected with different members of the CEACAM family.
  • Vectors encoding the full-length version of human CEACAM 1, 3, 4, 5, 6, 7, 8, 18, 19, 20 and 21 and 20 are used to express these proteins at the surface of PEAK and/or CHO cells as described in Example 2.
  • Non-transfected PEAK and/or CHO cells are used as negative control. Cells are harvested, counted, checked for viability and resuspended at 3 ⁇ 10 6 cells/ml in FACS buffer (PBS 2% BSA, 0.1% NaN 3 ).
  • An antibody according to the invention is found as non-binding to said CEACAM, if no bound antibody is detected by the PE-conjugated anti-human IgG Fc secondary antibody.
  • Biotinylated recombinant human CEACAM5 or CEACAM6 proteins or recombinant cynomolgus monkey CEACAM5 are captured at 0.5 ⁇ g/mL in a streptavidin coated 96-well microplate.
  • the plate is washed and monoclonal anti-CEA bivalent antibodies of the present invention are added as a broad concentration-range (e.g. from 5x10-4 to 10 ⁇ g/mL) and incubated during 1 hr.
  • the plate is washed and bound antibodies are detected with an anti-human IgG(Fc)-HRP (Jackson ImmunoResearch). After washing, the plate is revealed with Amplex Red reagent (Molecular Probes).
  • Antibody name EC50 (nM) binding to human CEACAM5 EC50 (nM) binding to cyno CEACAM5 AC41 0.3 0.6 AC42 0.1 0.6 AC43 0.08 0.7 AC44 0.09 0.07 AC45 0.1 0.03 AC46 0.07 0.04 AC47 0.03 0.06 AC48 0.05 0.04 AC49 0.1 0.03 AC50 0.09 0.05 AC52 0.03 0.02 AC53 0.03 0.02 AC54 0.03 0.02 AC55 0.03 0.02 AC56 0.02 0.02 AC57 0.01 0.02 AC58 0.04 0.04 AC59 0.05 0.05 AC60 0.013 0.06 AC61 0.008 0.02 AC62 0.012 0.01 AC63 0.016 0.02 AC64 0.016 0.03 AC65 0.014 0.03 AC66 0.012 0.01
  • the simultaneous expression of one heavy chain and two lights chain in the same cell can lead to the assembly of three different antibodies. Simultaneous expression can be achieved in different ways such as that the transfection of multiple vectors expressing one of the chains to be co-expressed or by using vectors that drive multiple gene expression.
  • the vector encoding the different anti-CEACAM5 antibodies are co-transfected with another vector expressing the heavy and light chain of anti-CD47 antibody K2 (SEQ ID NO:5 and 11), an anti-CD47 antibody bearing the same common heavy chain and that is described in US 2014/0303354 .
  • the two light chains are cloned into the vector pNovi ⁇ H ⁇ that is previously generated to allow for the co-expression of one heavy chain, one Kappa light chain and one Lambda light chain as described in US 2012/0184716 and WO 2012/023053 , each of which is hereby incorporated by reference in its entirety.
  • the expression of the three genes is driven by human cytomegalovirus promoters (hCMV) and the vector also contains a glutamine synthetase gene (GS) that enables the selection and establishment of stable cell lines.
  • the common VH and the VL genes of the anti- CEACAM5 IgG and of the anti-CD47 IgG are cloned in the vector pNovi ⁇ H ⁇ , for transient expression in mammalian cells. Peak cells are cultured in appropriate Flask with suitable cells number and culture medium volume (containing fetal bovine serum). Plasmid DNA is transfected into the cells using Lipofectamine 2000) according to manufacturer's instructions. Antibody concentration in the supernatant of transfected cells is measured during the production using OctetRED96. According to antibody concentration, supernatants are harvested 5 to 7 days after transfection and clarified by centrifugation at 1300 g for 10 min. The purification process is composed of three affinity steps.
  • the FcXL affinity matrix (Thermofisher Scientific) is washed with PBS and then added in the clarified supernatant. After incubation overnight at +4°C, supernatants are centrifuged at 2000 g for 10 min, flow through is stored and resin washed twice with PBS. Then, the resin is transferred on Amicon Pro columns and a solution containing 50 mM glycine at pH 3.0is used for elution. Several elution fractions are generated, pooled and desalted against PBS using 50 kDa AmiconTM Ultra Centrifugal filter units (Merck KGaA, Darmstadt, Germany).
  • the elueted product containing total human IgGs from the supernatant, is quantified using a Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, Del.) and incubated for 15 min at RT and 20 rpm with the appropriate volume of Kappa select affinity matrix (GE Healthcare). Incubation, resin recovery, elution and desalting steps are performed as described previously. The last affinity purification step is performed using the lambda Fab select affinity matrix (GE Healthcare) applying the same process as for the two previous purifications. The final product is quantified using the Nanodrop. Purified bispecific antibodies are analyzed by electrophoresis in denaturing and reducing conditions.
  • the Agilent 2100 Bioanalyzer is used with the Protein 80 kit as described by the manufacturer (Agilent Technologies, Santa Clara, Calif., USA). 4 ⁇ L of purified samples are mixed with sample buffer supplemented with dithiothreitol (DTT; Sigma Aldrich, St. Louis, Mo.). Samples are heated at 95°C for 5 min and then loaded on the chip. All samples are tested for endotoxin contamination using the Limulus Amebocyte Lysate test (LAL; Charles River Laboratories, Wilmington, Mass.).
  • Dual-targeting bispecific antibodies bind to two different antigens on the surface of the same cell.
  • a dual targeting ⁇ antibody composed of one arm binding with high affinity to CEACAM5 or to CEACAM5 and CEACAM6, and a second arm binding with lower affinity to CD47-but sufficient to inhibit CD47/SIRP ⁇ upon CEACAM5 or CEACAM5 and CEACAM6 co-engagement with CD47 should allow preferential inhibition of CD47 in cancer versus normal cells.
  • the binding affinity of the antibodies according to the invention to human CD47 can be evaluated by surface plasmon resonance technology using a Biacore T200 instrument.
  • the biotinylated human CD47 soluble recombinant protein can be captured on a streptavidin coated sensor chip (Series S Sensor Chip SA). Then a concentration series of the test antibody can be injected over the surface, with regeneration of the surface between each injection.
  • Such measurements were performed with a CD19xCD47 ⁇ bispecific antibody.
  • the binding affinity measured in repeated determinations was between 400 and 500 nM.
  • the CD47 binding arm of this antibody is the same as the CD47 binding arm of the CEAxCD47 bispecific antibodies of this invention (in one embodiment the sequences SEQ ID NO:1 to 14, 119, 120 and 181 of table 2, sequence list). According to the knowledge of the inventors that same experiments performed with the CEAxCD47 bispecific antibodies of the invention will provide similar results within the standard deviation of such experiments. This hold especially also true for the reference bispecific antibody K2AC54.
  • MKN45 cancer cells expressing both CD47 and CEACAM5, are stained with CFSE violet to allow the imaging system (CX5) to detect the cells.
  • CX5 imaging system
  • 3'000 stained MKN45 cells per well are seeded in a 384 optical well plate (Costar) and incubated for 50 minutes with increased concentrations of bispecific antibodies of the invention (1.9 pM to 333 nM, in quadruplicates). Then, a fixe concentration of SIRP ⁇ mouseFc premixed with anti-mouse IgG-Fc AF647 coupled antibody (Jackson Immunoresearch diluted 1:2000) is added at 50ng/mL final.
  • Table 4 shows the potency of several CEAxCD47 bispecific antibodies at inhibiting CD47/SIRP ⁇ binding displaying a range of IC50, from 0.16nM to 9.4nM.
  • Epitope binning is a competitive immunoassay used to characterize the binding of antibodies according to the invention or e.g. the binding of the related anti-CEA (target protein) antibodies of the first binding part.
  • a competitive blocking profile of an antibody binding to the target protein is created against antibodies also binding to this target protein and for which the binding epitope has already been established/published. Competition to one of these reference antibodies indicate that the antibody has the same or a closely located epitope and they are "binned" together.
  • CEACAM5 mAbs which are part of the bispecific antibodies of the present invention to compete with CEACAM5 reference antibodies is tested by ELISA on recombinant human CEACAM5 with the following reference antibodies carrying a mouse Fc region: SM3E, sequences of mAb derived from SM3E described in patent US20050147614A1 , mAb produced using standard methods; MEDI, mAb derived from MEDI-565 described in patent WO2016036678A1 ; SAR, mAb derived from Mab2_VLg5VHg2 described in patent EP3199552A1 ; CH1A1A, mAb derived from CH1A1A-2F1 described in patent US20120251529 and by Klein et al in Oncoimmunology, 2017 Jan 11;6(3 ); humanized T84.66 mAb derived from variant 1 described in patent WO2017055389 ; LAB mAb derived from hMN14 described
  • Biotinylated human CEACAM5 is coated at 0.5 ⁇ g/ml in a Streptavidin-coated 96-well plate and incubated with 10 ⁇ g/ml of the reference mAbs or an irrelevant mAb carrying a mouse Fc region for 1 hour.
  • the CEACAM5 mAbs (as bivalent monoclonal anti-CEA antibodies and not as respective CEAxCD47 bispecific antibodies) are added at 0.2 ⁇ g/ml for 1 hour at room temperature.
  • the plate is washed and the bound CEACAM5 mAbs are detected with an anti-human IgG(Fc)-HRP (Jackson ImmunoResearch). After washing, the plate is revealed with Amplex Red reagent.
  • the fluorescence signal is measured on a Synergy HT plate reader (Biotek).
  • the competition experiments were for all of the CEAxCD47 bispecific antibodies according to the invention performed with the respective anti-CEA bivalent monoclonal antibodies.
  • binding of such a monoclonal antibody to CEACAM5 was reduced by the respective tool antibody by 80% or more, it was concluded that the CEAxCD47 bispecific antibody is classified to bind competitively with the tool antibody.
  • a CEAxCD47 antibody is identified as non-competitive with a tool antibody in case binding of the respective anti-CEA bivalent mAb to CEACAM5 is reduced by 20% or less if the results with and w/o addition of a tool antibody are compared.
  • AC58, AC59 and the bispecifics antibodies K2AC58 and K2AC59 resulting from a Lead Optimization approach as described in Example 5 (2.) of parenteral antibody AC21 (identified in a phage display library approach), are much weaker in binding to MKN-45 cells and in phagocytosis of MKN-45 cells (see table 4 - low Emax for binding and figure 7 for phagocytosis compared to reference biAb K2AC54) as compared to the SM3E binding anti CEA antibodies according to the invention and the respective SM3E binding bispecific antibodies according to the invention.
  • K2AC41 means that there is a fully kappa LC (VL and CL as kappa) in the K2 anti CD47 arm and in the AC41 arm the VL is kappa and therefore a lambda hybrid CL is introduced (or in other words AC41 LC is hybrid).
  • VL and CL as kappa
  • CL lambda hybrid
  • AC41K2 H-CL1 the AC41 LC is fully kappa, therefore in the CD47 arm the LC is hybrid with kappa VL and lambda CL (same for K2AC42 and K2AC43 and also the CEAxCD47 bispecific antibodies based on AC60 to AC66).
  • Biotinylated recombinant human CEACAM5 or CEACAM6 proteins are captured at 0.5 ⁇ g/mL in a streptavidin coated 96-well microplate.
  • the plate is washed and monoclonal anti-CEA bivalent antibodies of the present invention are added as a broad concentration-range (e.g. from 5x10 -4 to 1 ⁇ g/mL) and incubated during 1 hr.
  • the plate is washed and bound antibodies are detected with an anti-human IgG(Fc)-HRP (Jackson ImmunoResearch). After washing, the plate is revealed with Amplex Red reagent (Molecular Probes). The fluorescence signal is measured on a Synergy HT plate reader (Biotek).
  • results obtained for the monoclonal antibodies 49 and AC50 are contained in table 6 and figures 4 and 5 ; these antibodies show balanced CEACAM5 and CEACAM6 binding, that means EC50 for binding to CEACAM5 and CEACAM6 are similar (range of the ratio of the EC50 for CEACAM5 binding to CEACAM6 binding of balanced antibodies from 0.2 to 5). Antibodies with a ratio outside such ranges are considered as not balanced. Table 6. EC50 binding on human CEACAM5 and human CEACAM6 by ELISA using recombinant proteins for two anti-CEA mAbs. Antibody name EC50 (nM) binding to human CEACAM5 EC50 (nM) binding to human CEACAM6 AC49 0.1 0.2 AC50 0.09 0.03
  • ADCP and ADCC mediated by CEAxCD47 bispecific antibodies is CEA dependent
  • healthy PBMC were activated overnight at 37°C with RPMI/10% heat inactivated FCS supplemented with 10 ng/mL of recombinant hIL-2.
  • targets cells i.e. cancer cells expressing the CEACAM5
  • cells were incubated with 100 ⁇ Ci Cr51 (Perkin Elmer, 37°C, 1h). After washing, cells were opsonized with test antibodies (30 min, 37°C). Cr51-loaded cancer cells were then mixed with PBMC cells to obtain the final 80:1 or 50:1 ratio between effector (PBMC) and target cells (CEACAM5-expressing cells). The cell mixture was incubated for 4h at 37°C before being centrifuged for 10 min at 1500 rpm.
  • % specific ADCC ((sample counts per minute (cpm) - nonspecific lysis control cpm)/(total lysis control cpm - negative control cpm)) x 100%.
  • ADCC of the CEAxCD47 bispecific antibodies was tested in the following assay: Healthy PBMC were activated overnight at 37°C with RPMI/10% heat inactivated FCS supplemented with 10 ng/mL of recombinant hIL-2. The next day, target cells (e.g. MKN45 cancer cells) are opsonized with different concentrations of tested antibodies. The PBMCs and the opsonized target cells are co-incubated at a ratio effector/target 50/1 in round bottom plates for 6 hours at 37°c in a cell culture incubator.
  • target cells e.g. MKN45 cancer cells
  • the percentage of phagocytosis (representing the percentage of macrophages which have engulfed at least one tumor cell) is determined.
  • the imaging-based method which makes use of the CellInsight CX5 High Content Screening Platform, the phagocytosis index, defined as the average number of target cells engulfed by 100 macrophages, is determined (see figure 3 ).
  • Phagocytosis Assays 1. Imaging assay based on CellInsight CX5 High Content Screening Platform and 2. Flow cytometry based assay
  • PBMCs Human peripheral blood mononuclear cells
  • M-CSF human macrophage colony-stimulating factor
  • Non-adherent cells are subsequently eliminated in the differentiation phase (day+1) by exchanging the cell culture medium, and adherent cells representing macrophages are detached using cell dissociation buffer (Sigma-Aldrich) and washed in complete medium the day of use (day8 or day9) for ADCP experiment based on cytometry.
  • cell dissociation buffer Sigma-Aldrich
  • Macrophages (stained with calcein red orange) adhering to microplate wells are co-incubated with Calcein AM-labeled target tumor cells at an effector: target cells ratio of 1:3 for 2.5 hours at 37 degree C in the presence of different concentrations of the to be tested antibody.
  • supernatants are replaced by complete culture medium and the microplates are imaged with the CellInsightTM CX5 High Content Screening Platform. 1500 macrophages are acquired and analyzed per well. Phagocytosis is evidenced as double-positive events (macrophage + target tumor cell) and the phagocytosis indexes are calculated by the CellInsightTM manufacturers' software.
  • ADCP phagocytosis
  • ADCP can also be measured by a method as described as follows:
  • the macrophages are co-incubated with CSFE-labeled target tumor cells (e.g. MKN-45, LS174T or HPAC tumor cells) at an effector: target cells ratio of e.g. 3:1 for 2.5 hours at 37 degree C in the presence of different concentrations of to be tested antibody.
  • target tumor cells e.g. MKN-45, LS174T or HPAC tumor cells
  • target cells ratio of e.g. 3:1 for 2.5 hours at 37 degree C in the presence of different concentrations of to be tested antibody At the end of the incubation period, biotinylated anti-human CD14 antibody and Strep-Cy5 are added to label the macrophages.
  • the cells are then washed and subjected to flow cytometry analysis. Phagocytosis is evidenced by double-positive events CD14+ and CFSE+. Percentage of phagocytosis is presented as the ratio between CD14+/
  • Example 10 Binding of CEAxCD47 bispecific antibodies of the invention to MNK-45 cells; measurement of competition of binding with CEAxCD3 bispecific antibodies
  • CD47xCEACAM5 bispecific antibody is tested on e.g. CEA-expressing human gastric adenocarcinoma cells (MKN-45, DSMZ ACC 409).
  • Cells are harvested, counted, checked for viability and resuspended at 3 ⁇ 10 6 cells/ml in FACS buffer (PBS 2% BSA, 0.1% NaN3). 100 ⁇ l of the cell suspension are distributed in V-bottom 96-well plates (3 ⁇ 10 5 cells/well). The supernatant is removed by centrifugation 3 minutes at 4°C, 1300 rpm. Increasing concentrations of the antibody according to the invention are then added into the wells and incubated for 15 minutes at 4°C.
  • FACS buffer PBS 2% BSA, 0.1% NaN3
  • CEAxCD3 T-cell bispecific antibodies like CEA-TCB or CEA-TCB1
  • the binding of the CEACAM5xCD47 to MKN-45 cells can be determined as described above, but with and w/o addition of the CEAxCD3 T-cell bispecific antibody to study if a CEAxCD3 T-cell bispecific antibody as combination partner for the CEAxCD47 bispecific antibodies of this invention is competitive for binding to CEA or not.
  • Example 11 Production and Purification of fucosylated and afucosylated bispecific antibodies, e.g. K2AC50 and K2AC54
  • a CHO pool (one for K2AC50 and one for K2AC54) is inoculated at a viable cell concentration of 0.3 x 10 6 cells/mL in a Thomson erlen device with a working volume of 700 mL or 100 mL for the production of fucosylated and afucosylated antibodies, respectively. All the pools are operated in a 15 days duration fed-batch mode using CDACF medium CDCHO and an adapted feeding regime.
  • bolus of 200 ⁇ M fucose inhibitor (1,3,4-Tri-O-acetyl-2-deoxy-2-fluoro-L-fucose) are added at day 0, 5, 8 and 11 during the fed batch process based on afucosylation strategy described by Rillahan et al. Nature Chem. Biol. 2012 Jul;8(7):661-8 and based on EP2282773 .
  • Harvest of the K2AC50 AND K2AC54 pools supernatants containing fucosylated or afucosylated antibodies is performed after 15 days of Fed batch culture.
  • Harvests of CHO pools supernatants are clarified using the Sartoclear Dynamics® Lab V Cell Harvesting Sartorius system (see supplier instructions).
  • purification of fucosylated and afucosylated bispecific antibodies according to the invention is a three affinity step purification process.
  • antibody concentration in the supernatant of bispecific antibody pools is measured using OctetRED96 in order to use columns with appropriate volume of affinity matrix.
  • Each clarified CHO pool supernatant containing fucosylated or afucosylated bispecific antibodies is loaded onto a MabSelect SuRe (MSS) column (GE Healthcare) without prior adjustment, to remove a major part of cell culture contaminants.
  • MSS eluate is then treated by low pH hold to inactivate viruses, and neutralized at pH 6 with Tris 1M pH9.
  • the MSS eluate's is then loaded onto the LambdaFabSelect (LFS) column (GE Healthcare) to remove monospecific ⁇ (mono ⁇ ).
  • the LFS eluate is then pH adjusted at pH 6.
  • the LFS is loaded onto the Capto L (CL) column (GE Healthcare) to remove monospecific ⁇ (mono ⁇ ).
  • the CL Eluate is pH adjusted before storage.
  • the final material is then concentrated and diafiltered into the final formulation buffer, its concentration adjusted using the Nanodrop. Fucosylated and afucosylated
  • Bispecific antibodies according to the invention will be aliquoted and stored at -80°C until delivery.
  • Purified bispecific antibodies are analyzed for sizing by electrophoresis in denaturing and reducing conditions with the Agilent 2100 Bioanalyzer using the Protein 80 kit as described by the manufacturer (Agilent Technologies, Santa Clara, Calif., USA). Aggregation level is assessed by size exclusion chromatography (SEC-UPLC) using the ACQUITY UPLC H-Class Bio System (Waters).
  • Charge variant analysis of purified bispecific antibodies is achieved by isoelectric focusing technique ( IEF ) using the Multiphor II Electrophoresis System (GE Healthcare).
  • N -linked complex biantennary glycoforms of fucosylated and afucosylated antibodies will be determined using the throughput microchip-CE method on the LabChip GXII Touch (Perkin Elmer). All antibodies are tested for endotoxin contamination using the Limulus Amebocyte Lysate test (LAL; Charles River Laboratories, Wilmington, Mass). Typical afucosylation achieved by this method is expected to be in the range of 70 to 90%.
  • afucosylated bispecific antibodies according to the invention can be produced also according to the method as follows: Material and Methods are according to Naoko Yamane-Ohnuki et al., Biotech. Bioeng.; 87 (2004) 614-622 .
  • RNA is isolated from CHO/DG44 cells using the RNeasy® Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed with oligo-dT using a Superscript first-strand synthesis system for reverse transcript-polymerase chain reaction (RT-PCR) (Invitrogen, Carlsbad, CA).
  • RNeasy® Mini Kit Qiagen, Hilden, Germany
  • RT-PCR reverse transcript-polymerase chain reaction
  • a Chinese hamster FUT8 cDNA is amplified from single-stranded CHO/DG44 cell cDNAs by PCR using primers 5V-GTCTGAAGCATTATGTGTTGAAGC-3V (SEQ ID NO:187) and 5V-GTGAGTACATTCATTGTACTGTG-3V (SEQ ID NO:188), designed from the murine FUT8 cDNA ( Hayashi, 2000; DNA Seq 11:91-96 ).
  • the targeted disruption of the FUT8 gene in CHO/DG44 cells is carried out using two replacement vectors, pKOFUT8Neo and pKOFUT8Puro.
  • the 9.0-kb fragment of the FUT8 gene including the first coding exon is isolated by screening the CHO-K1 cell E-genomic library (Stratagene, La Jolla, CA) with the Chinese hamster FUT8 cDNA as a probe to establish the targeting constructs.
  • a 234-bp segment containing the translation initiation site is replaced with the neomycin-resistance gene (Neor) cassette or the puromycin-resistance gene (Puror) cassette from plasmid pKOSelectNeo or pKOSelectPuro (Lexicon, TX), respectively, flanked by loxP sites.
  • the diphtheria toxin gene (DT) cassette from plasmid pKOSelectDT (Lexicon) is inserted at the 5V homologous region.
  • the resulting targeting constructs, pKOFUT8Neo and pKOFUT8Puro included the 1.5-kb 5V homologous sequence and the 5.3-kb 3V homologous sequence. Before transfection, the targeting constructs are linearized at a unique SalI site.
  • Subconfluent CHO/DG44 cells (1.6 106) are electroporated with 4 Ag of linearized pKOFUT8Neo at 350 V and 250 AF using a Bio-Rad GenePulser® II. After electroporation, transfectants are selected with 600 Ag/mL G418 (Nacalai Tesque, Kyoto, Japan). Genomic PCR is performed in 96-well plates by the modified microextraction method reported previously ( Ramirez-Solis et al., 1992; Anal Biochem 201:331- 335 .) using the following primers:
  • Homologous recombinants are identified by the 1.7-kb fragment obtained using genomic PCR and confirmed by Southern blot analysis using the 221-bp fragment amplified with the following primers:
  • the hemizygous clone is subject to a second round of homologous recombination using linearized pKOFUT8Puro and drug selection with 15 Ag/mL puromycin (Sigma-Aldrich, St. Louis, MO) as described earlier.
  • the identified homozygous disruptants are electroporated with the Crerecombinase expression vector pBS185 (Invitrogen) to remove drug-resistance gene cassettes from both FUT8 alleles.
  • FUT8(-) cell lines are electroporated with an expression vector encoding an bispecific antibody according to the invention and selected in media lacking hypoxanthine and thymidine.
  • the confluent transfectants are cultured in Ex-Cell® 301 Medium (JRH Biosciences, Lenexa, KS) for 1 week.
  • the antibody is purified from culture supernatants using MabSelectTM (Amersham Biosciences, Piscataway, NJ). Further purification steps can be anion/cation exchange chromatography, size exclusion chromatography and especially purification using kappa respectively lambda selective resins as described above.
  • afucosylated bispecific antibodies of the invention can be produced also according to the method/technology as follows and described in, US8642292 .
  • This technology is designed to configure the stable integration of a heterologous bacterial enzyme into an antibody producer cell line like a CHO cell line or others. By this, the de novo synthesis of fucuse from D-mannose is blocked. If in addition production cells are cultivated in fucose free medium, as a result antibodies with a stable level of afucosylation are produced.
  • fucose is generated through two routes
  • the salvage pathway can be completely blocked by omission of fucose from the culture medium.
  • the de novo biosynthesis pathway can be blocked by converting the intermediate GDP-4-keto-6-deoxy-D-mannose of this pathway to GDP-D-rhamnose instead of GDP-4-keto-6-deoxy-D-galactose. This is achieved by bringing the bacterial enzyme GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD) into the production cell line, respectively by stable integration of the gene encoding for RMD into the production cell line. Even rather low amounts of RMD expressed in the production cell line completely block the de novo synthesis pathway of the production cell.
  • RMD GDP-6-deoxy-D-lyxo-4-hexulose reductase
  • This technology will be used to construct production cell lines, e.g. CHO based cell lines, designed for the production of afucosylated antibodies of the invention as well as to existing production cell lines which already produce antibodies of the invention and are engineered to produce the antibodies with fucose content reduced by 80% to 100%.
  • production cell lines e.g. CHO based cell lines
  • the anti-tumor activity of a bispecific antibody according to the invention can be evaluated in Xenograft models, e.g. by the following model: 1 to 3x10 6 CEA positive tumor cells like MKN-45, LS174T, or SNUC-1 cells are implanted subcutaneously in e.g. NOD/SCID mice. Tumor volumes are measured 3 times per week. After 3 or 5 or 7 or 9 days after the tumor cell implantation or alternatively when the tumor graft reached a volume of approx. 100 to 300mm 3 , mice are randomized into groups (e.g. 4 to 6 mice per group) and the antibody treatment is initiated. This experiment could e.g.
  • Antibody is injected e.g. i.v. every week until the end of the experiment (approximately d25).
  • Antibodies are administered at e.g. daily or 3 times a week or weekly etc doses of e.g. 1 or 2.5 or 5 or 10 or 20 mg/kg.
  • Combinations of a bispecific antibody of this invention with a CEAxCD3 bispecific antibody can be tested in an appropriate model.
  • Models, in which the combination of an antibody according to the invention together with CEA-TCB or CEA-TCB1 can be tested are e.g. described by Bacac et al (Clin. Cancer Res., 22(13);3286-97;2016 ) and are also used, especially for combination studies of CEACAM5xCD47 or CEACAM5/6xCD47 and CEA-TCB or CEA-TCB 1.
  • Usually human PBMC have to be engrafted in such a model.
  • Example 14 Cytokine release tested in whole blood and PBMCs from healthy human donors human blood
  • an in vitro cytokine release assay can be performed using whole blood (WB CRA) with minimal dilution by the test antibodies (95% v/v blood) in aqueous presentation.
  • This assay format is considered to mimic more closely the in vivo environment, containing factors at physiological concentrations that may influence mechanisms of cytokine release.
  • this format is thought to be poorly predictive of T cell-mediated cytokine release (e.g., anti-CD28).
  • the assay can be also performed using peripheral blood mononuclear cells (PBMCs) from healthy human donors and with an immobilized mAb (Solid Phase, SP) presentation to assess T cell-mediated cytokine release (PBMC SP CRA).
  • PBMC SP CRA peripheral blood mononuclear cells
  • This assay format simulates cross-linking and high density presentation of mAbs, which may occur in vivo (e.g. clustering of the target via the interaction of the Fc part of the antibody with Fc ⁇ receptors on other immune cells or the cross-linking of mAbs by anti-drug antibodies).
  • This format is predictive of T cell-mediated cytokine release.
  • Example 15 Antibody Binding to Erythrocytes, Phagocytosis of Erythrocytes, and Platelet activation and aggregation
  • human whole blood samples collected from healthy donors in citrate can be mixed with 3 ⁇ g/mL of AF488-coupled CEA x CD47 bispecific antibodies of this invention, B6H12.2 or isotype control and surface staining antibodies (PE-Cy7 anti-hCD45 and PE anti-hCD41a, for platelets only) for 30 min at 4°C.
  • whole blood is divided in two samples: 5 ⁇ L are diluted and washed in PBS for erythrocyte analysis while 150 ⁇ L are incubated with erythrocyte lysing solution and washed for platelet analysis. Samples are acquired on a CytoFLEX instrument and analyzed with the FlowJo software to determine MFI values.
  • human red blood cells can be isolated from human whole blood by centrifugation at 300xg, washed twice in PBS, labeled with CFSE-(Carboxyfluorescein succinimidyl ester) and pre-incubated with the test antibody for 1 hour at 37° C before the addition of macrophages.
  • Labeled RBCs can be cultured with human macrophages in the presence of an antibody according to the invention or control (non-binding IgG1 antibody) for one hour at a target-to-effector ratio of 200:1. After culture, cells are stained with anti-CD14-APC and analyzed by flow cytometry.
  • Phagocytosis was quantitated as the percent of CD14+ events (macrophages) that are also CFSE+ and had therefore engulfed at least one RBC (events are gated on singlets). Phagocytosis and FACS analysis is done as described in example 9, except that the erythrocytes were lysed with FACS lysing solution after macrophage staining.
  • CEAxCD47 bispecific antibodies In a standard flow cytometry experiment the ability of CEAxCD47 bispecific antibodies to induce human platelet activation in whole blood of seven human healthy donors was measured by the upregulation of surface marker CD62P. Briefly, 5 ⁇ L of whole blood is incubated with 10 ⁇ L of each sample (prepared at 2X) for 15 minutes at room temperature. Each tested antibody is added at different concentrations (0, 0.02, 0.2, 2, 20 and 200 ⁇ g/mL). Adenosine diphosphate (ADP) and anti-CD9 (ALB6), included as positive control reagents known to induce platelet activation, are added at a concentration of 10 ⁇ M and 10 ⁇ g/mL, respectively.
  • ADP Adenosine diphosphate
  • ARB6 anti-CD9
  • the potential for aggregation in the presence of CD47/CEA bispecific antibody could be assessed on platelet rich plasma (PRP).
  • PRP platelet rich plasma
  • PRP is challenged with ADP at 10 ⁇ M and 5 ⁇ M or with the test articles at 200, 100, 20, 25, and 12.5 ⁇ g/mL, as well as with saline or the isotype control.
  • Platelet aggregation can be evaluated throughout platelet stimulation (i.e. 10 min) with a Thrombo-aggregometer TA 4V under constant stirring.
  • Thrombosoft 1.6 software SD Innovation, Frouard, France) can be used for analysis of the data.
  • cynomolgus monkey cross-reactive antibodies could be tested in vivo in Cynomolgus Monkeys for any effect on hematology parameters (including RBC and platelets).
  • An antibody according to the invention is e.g. given to cynomolgus monkeys per intravenous route, at doses up to 100 mg/kg, on a weekly basis.
  • Hematology parameters including red blood cell and platelet counts, are monitored over time and compared to control values in monkeys (pre-dose values). Hematology parameters are determined by routine methods.
  • Example 17 Determination of Pharmacokinetics properties in cynomolgus monkeys
  • Blood withdrawals are scheduled according to the experimental protocol at multiple time points, e,g, 0.25, 1, 4, 8, 24, 48, 72, 96, 120, 168, 240, 336, 504 (day 22), 672 (day 29), 840 (day 36), 1008 (day 43), 1176 (day 50) and 1344h (day 57) after the intravenous administration of the bispecific antibody.
  • Blood samples of approximately 2 mL per animal and time-point are collected. Concentrations of the antibodies were either measured in serum or in plasma. An ELISA test is developed and validated to measure the concentrations. Each sample is measured in duplicates.
  • PK parameters like Cmax, clearance, elimination half-life, area under the curve etc. can be determined by using industry standard software (Phoenix WinNonlin; non-compartmental analysis).
  • Example 18 ADCP Mediated by Bispecific Antibodies in presence of CEA-TCB and CEA-TCB1
  • calcein AM-labeled MKN45 cells used as target cells are pre incubated or not with a fixed dose of CEA-TCB (300nM) or CEA-TCB1 (30nM) for 20 min at RT. After this incubation different concentrations of tested antibody are added in appropriate well for 20 min. Then macrophages (stained with calcein red orange) adhering to microplate wells are co-incubated with the opsonized labeled target tumor cells at an effector:target cells ratio of 1:3 for 2.5 hours at 37 °C.
  • the ADCP is performed in a presence of 1mg/mL of human hIgG.
  • PBMCs peripheral blood mononuclear cells
  • Target cells (MKN45 engineered to express Luciferase) were opsonized with a combination of antibodies, i.e. with a CEAxCD3 T-cell bispecific antibody at certain concentrations together with certain concentrations of of aCEAxCD47 bispecific antibody.
  • Opsonized targets were added to the plates containing macrophages and autologous PBMCs; and the plates were incubated at 37°C for 48h. After 48h, half of the well medium was removed and a solution of 2X Luciferin was added to the plates to obtain a final concentration of 150 ⁇ g/mL. After 5 minutes incubation at RT, plates were read using a Synergy NEO. Percentage of viability was calculated dividing the luminescence value (minus background) by the control containing only target cells and multiplying by 100. Percentage of killing was then extrapolated by subtracting the percentage of viability to 100.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11753481B2 (en) 2020-12-18 2023-09-12 Lamkap Bio Beta Ltd Bispecific antibodies against CEACAM5 and CD47
WO2023186112A1 (fr) * 2022-04-02 2023-10-05 普米斯生物技术(珠海)有限公司 Anticorps multispécifique ciblant ceacam et cd3 et son utilisation

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* Cited by examiner, † Cited by third party
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US20240002544A1 (en) 2022-03-07 2024-01-04 Novimmune Sa Cd28 bispecific antibodies for targeted t cell activation
WO2023242351A1 (fr) 2022-06-16 2023-12-21 Lamkap Bio Beta Ag Polythérapie d'anticorps bispécifiques dirigés contre ceacam5 et cd47 et anticorps bispécifiques dirigés contre ceacam5 et cd3

Citations (98)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999054342A1 (fr) 1998-04-20 1999-10-28 Pablo Umana Modification par glycosylation d'anticorps aux fins d'amelioration de la cytotoxicite cellulaire dependant des anticorps
WO2000041474A2 (fr) 1999-01-15 2000-07-20 Novartis Ag Immunotoxines anti-cd3 et leurs utilisations therapeutiques
EP1176195A1 (fr) 1999-04-09 2002-01-30 Kyowa Hakko Kogyo Co., Ltd. Methode de regulation de l'activite d'une molecule immunologiquement fonctionnelle
US20020165360A1 (en) 2000-11-30 2002-11-07 Junghans Richard P. Chimeric effector cell receptors against carcinoembryonic antigen
US20030115614A1 (en) 2000-10-06 2003-06-19 Yutaka Kanda Antibody composition-producing cell
WO2003056914A1 (fr) 2001-12-27 2003-07-17 Glycofi, Inc. Procede d'ingenierie de structures de carbohydrates de type mammalien
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
US20030175884A1 (en) 2001-08-03 2003-09-18 Pablo Umana Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
WO2003084570A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Medicament contenant une composition d'anticorps appropriee au patient souffrant de polymorphisme fc$g(g)riiia
WO2003085119A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Procede d'amelioration de l'activite d'une composition d'anticorps de liaison avec le recepteur fc$g(g) iiia
WO2003099196A2 (fr) 2002-05-23 2003-12-04 Cure Tech Ltd. Anticorps monoclonaux humanises immunomodulateurs servant a traiter une maladie neoplasique ou une immunodeficience
WO2004024927A1 (fr) 2002-09-12 2004-03-25 Greenovation Biotech Gmbh Procede de production de proteines
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US20040110704A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells of which genome is modified
US20040110282A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost
US20040132140A1 (en) 2002-04-09 2004-07-08 Kyowa Hakko Kogyo Co., Ltd. Production process for antibody composition
WO2004057002A2 (fr) 2002-12-20 2004-07-08 Greenovation Biotech Gmbh Ameliorations apportees ou associees a la production de proteines
WO2004063351A2 (fr) 2003-01-09 2004-07-29 Macrogenics, Inc. Identification et elaboration d'anticorps avec des regions du variant fc et procedes d'utilisation associes
WO2004065540A2 (fr) 2003-01-22 2004-08-05 Glycart Biotechnology Ag Constructions hybrides et leur utilisation pour produire des anticorps presentant une affinite de liaison accrue pour le recepteur fc et fonction d'effecteur
WO2004099249A2 (fr) 2003-05-02 2004-11-18 Xencor, Inc. Variants fc optimises et leurs procedes de generation
WO2005018572A2 (fr) 2003-08-22 2005-03-03 Biogen Idec Ma Inc. Anticorps ameliores possedant une fonction d'effecteur modifiee et procedes de fabrication associes
WO2005018669A1 (fr) 2003-08-18 2005-03-03 Macrogenics, Inc. Anticorps specifiques des proteines fc?riib et procedes d'utilisation associes
WO2005056759A2 (fr) 2003-12-04 2005-06-23 Xencor, Inc. Procedes pour produire des proteines variantes presentant une plus grande concentration en chaine hote et compositions de celles-ci
US20050147614A1 (en) 2002-07-01 2005-07-07 Begent Richard J.H. Antibodies against tumor surface antigens
WO2005063815A2 (fr) 2003-11-12 2005-07-14 Biogen Idec Ma Inc. Variants de polypeptides de liaison au recepteur fc$g(g) et procede apparentes
WO2005092925A2 (fr) 2004-03-24 2005-10-06 Xencor, Inc. Variantes d'immunoglobuline a l'exterieur de la region fc
WO2005110474A2 (fr) 2004-05-10 2005-11-24 Macrogenics, Inc. ANTICORPS SPÉCIFIQUES FcϜRIIB HUMANISÉS ET MÉTHODES D’UTILISATION
WO2006019447A1 (fr) 2004-07-15 2006-02-23 Xencor, Inc. Variantes genetiques de fc optimisees
WO2006023420A2 (fr) 2004-08-16 2006-03-02 Medimmune, Inc. Antagonistes de l'integrine possedant une action cytotoxique a mediation cellulaire dependante de l'anticorps amelioree
WO2006047350A2 (fr) 2004-10-21 2006-05-04 Xencor, Inc. Variants d'immunoglobuline igg a fonction effectrice optimisee
WO2006085967A2 (fr) 2004-07-09 2006-08-17 Xencor, Inc. Anticorps monoclonaux optimises anti-cd20 a variants fc
WO2006105338A2 (fr) 2005-03-31 2006-10-05 Xencor, Inc. Variants fc presentant des proprietes optimisees
WO2006116260A2 (fr) 2005-04-26 2006-11-02 Medimmune, Inc. Modulation de la fonction d'effecteur d'anticorps par ingenierie de domaine 'charniere'
WO2007005874A2 (fr) 2005-07-01 2007-01-11 Medarex, Inc. Anticorps monoclonaux humains diriges contre un ligand de mort programmee de type 1(pd-l1)
WO2007008943A2 (fr) 2005-07-08 2007-01-18 Xencor, Inc. Proteines optimisees qui ciblent la molecule ep-cam
WO2007021841A2 (fr) 2005-08-10 2007-02-22 Macrogenics, Inc. Identification et ingenierie d'anticorps presentant des zones de variants fc et procedes d'utilisation de ces anticorps
WO2007024249A2 (fr) 2004-11-10 2007-03-01 Macrogenics, Inc. Fonction effectrice obtenue par creation par genie biologique de regions d'anticorps fc
WO2007041635A2 (fr) 2005-10-03 2007-04-12 Xencor, Inc. Variants de fc dotés de propriétés de liaison aux récepteurs fc optimisées
WO2007042261A2 (fr) 2005-10-11 2007-04-19 Micromet Ag Compositions comportant des anticorps specifiques d'especes croisees et leurs utilisations
WO2007044616A2 (fr) 2005-10-06 2007-04-19 Xencor, Inc. Anticorps anti-cd30 optimises
WO2007048077A2 (fr) 2005-10-21 2007-04-26 Gtc Biotherapeutics, Inc. Anticorps ayant une meilleure activite cytotoxique cellulaire dependant des anticorps et leurs procedes de production et leur utilisation
WO2007071426A1 (fr) 2005-12-21 2007-06-28 Micromet Ag Préparations pharmaceutiques résistantes à l’ace soluble
WO2007106707A2 (fr) 2006-03-10 2007-09-20 Macrogenics, Inc. Identification et modification génétique d'anticorps avec des chaînes lourdes de variants et leurs procédés d'utilisation
WO2008015671A2 (fr) 2006-07-30 2008-02-07 Assaf Klar Procédé de conception et d'application d'une canalisation muticouche à flexibilité longitudinale améliorée
WO2008022152A2 (fr) 2006-08-14 2008-02-21 Xencor, Inc. Anticorps optimisés ciblant cd19
WO2008036688A2 (fr) 2006-09-18 2008-03-27 Xencor, Inc. Anticorps optimisés ciblant l'antigène hm1.24
WO2008091798A2 (fr) 2007-01-22 2008-07-31 Xencor, Inc. Anticorps de ca9 optimises et methodes d'utilisation associees
WO2008092117A2 (fr) 2007-01-25 2008-07-31 Xencor, Inc. Nouvelles insertions, délétions et substitutions d'immunoglobulines
WO2008091954A2 (fr) 2007-01-23 2008-07-31 Xencor, Inc. Anticorps cd40 optimisés et leurs procédés d'utilisation
WO2008098115A2 (fr) 2007-02-07 2008-08-14 Xencor, Inc. Anti-corps igf-1r optimisés et procédés utilisant ceux-ci
WO2008119567A2 (fr) 2007-04-03 2008-10-09 Micromet Ag Domaine de liaison spécifique d'espèces croisées
WO2008119566A2 (fr) 2007-04-03 2008-10-09 Micromet Ag Éléments de liaison bispécifiques spécifiques d'espèces croisées
WO2008121160A2 (fr) 2006-11-21 2008-10-09 Xencor, Inc. Anticorps optimisés qui ciblent cd5
WO2008119565A2 (fr) 2007-04-03 2008-10-09 Micromet Ag Domaine de liaison spécifique d'espèces croisées
WO2008140603A2 (fr) 2006-12-08 2008-11-20 Macrogenics, Inc. MÉTHODES POUR LE TRAITEMENT DE MALADIE AU MOYEN D'IMMUNOGLOBULINES COMPRENANT DES RÉGIONS FC QUI PRÉSENTENT DES AFFINITÉS ALTÉRÉES POUR FCγR D'ACTIVATION ET FCγR D'INHIBITION
WO2008150494A1 (fr) 2007-05-30 2008-12-11 Xencor, Inc. Procédés et compositions permettant l'inhibition de cellules d'expression du cd32b
WO2008156712A1 (fr) 2007-06-18 2008-12-24 N. V. Organon Anticorps dirigés contre le récepteur humain de mort programmée pd-1
US7521051B2 (en) 2002-12-23 2009-04-21 Medimmune Limited Methods of upmodulating adaptive immune response using anti-PD-1 antibodies
WO2009101611A1 (fr) 2008-02-11 2009-08-20 Curetech Ltd. Anticorps monoclonaux pour le traitement de tumeurs
WO2009114335A2 (fr) 2008-03-12 2009-09-17 Merck & Co., Inc. Protéines de liaison avec pd-1
WO2010027423A2 (fr) 2008-08-25 2010-03-11 Amplimmune, Inc. Compositions d'antagonistes de pd-1 et methodes d'utilisation associees
WO2010027827A2 (fr) 2008-08-25 2010-03-11 Amplimmune, Inc. Polypeptides co-stimulateurs ciblés et leurs procédés d'utilisation dans le traitement du cancer
WO2010033736A1 (fr) 2008-09-17 2010-03-25 Xencor, Inc. Nouvelles compositions et nouvelles méthodes de traitement d'affections médiées par les ige
WO2010037836A2 (fr) 2008-10-01 2010-04-08 Micromet Ag Anticorps monocaténaire bispécifique psmaxcd3, spécifique d'espèces croisées
WO2010037838A2 (fr) 2008-10-01 2010-04-08 Micromet Ag Anticorps monocaténaire bispécifique à domaine unique, spécifique d'espèces croisées
WO2010037837A2 (fr) 2008-10-01 2010-04-08 Micromet Ag Anticorps monocaténaires bispécifiques spécifiques d'antigènes cibles de masse moléculaire élevée
WO2010077634A1 (fr) 2008-12-09 2010-07-08 Genentech, Inc. Anticorps anti-pd-l1 et leur utilisation pour améliorer la fonction des lymphocytes t
EP2282773A2 (fr) 2008-05-02 2011-02-16 Seattle Genetics, Inc. Procédé et compositions pour préparer des anticorps et des dérivés d'anticorps avec une fucosylation centrale réduite
US20110064653A1 (en) 2009-09-16 2011-03-17 Immunomedics, Inc. Class I Anti-CEA Antibodies and Uses Thereof
US8008449B2 (en) 2005-05-09 2011-08-30 Medarex, Inc. Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics
WO2012023053A2 (fr) 2010-08-16 2012-02-23 Novimmune S.A. Procédé de production d'anticorps multispécifiques et multivalents
WO2012117002A1 (fr) 2011-03-02 2012-09-07 Roche Glycart Ag Anticorps anti-cea
WO2013012414A1 (fr) 2011-07-18 2013-01-24 Medimmune, Llc Régimes posologiques pour le traitement cancers exprimant cea
WO2013019906A1 (fr) 2011-08-01 2013-02-07 Genentech, Inc. Procédés de traitement du cancer à l'aide d'antagonistes se liant à l'axe pd-1 et inhibiteurs de mek
WO2013088259A2 (fr) 2011-10-19 2013-06-20 Novimmune S.A. Procédés de purification d'anticorps
WO2013173223A1 (fr) 2012-05-15 2013-11-21 Bristol-Myers Squibb Company Immunothérapie anticancéreuse par rupture de la signalisation pd-1/pd-l1
US8642292B2 (en) 2009-09-22 2014-02-04 Probiogen Ag Process for producing molecules containing specialized glycan structures
US20140140989A1 (en) 2012-02-06 2014-05-22 Inhibrx Llc Non-Platelet Depleting and Non-Red Blood Cell Depleting CD47 Antibodies and Methods of Use Thereof
WO2014087248A2 (fr) 2012-12-03 2014-06-12 Novimmune S.A. Anticorps anti-cd47 et leurs procédés d'utilisation
WO2014113510A1 (fr) 2013-01-15 2014-07-24 Xencor, Inc. Elimination rapide de complexes antigéniques à l'aide de nouveaux anticorps
US20140242079A1 (en) 2013-02-26 2014-08-28 Roche Glycart Ag Bispecific t cell activating antigen binding molecules
WO2015026634A1 (fr) 2013-08-20 2015-02-26 Merck Sharp & Dohme Corp. Traitement du cancer avec une combinaison d'un antagoniste de pd-1 et du dinaciclib
WO2015112534A2 (fr) 2014-01-21 2015-07-30 Medimmune, Llc Compositions et procédés pour moduler et réorienter des réponses immunitaires
WO2016007235A1 (fr) 2014-07-11 2016-01-14 Genentech, Inc. Anticorps anti-pd-l1 et leurs utilisations
WO2016036678A1 (fr) 2014-09-02 2016-03-10 Medimmune, Llc Formulations d'anticorps bispécifiques
US20160144009A1 (en) 2013-04-29 2016-05-26 The Board Of Trustees Of The Leland Stanford Junior University Use of Anti-CD47 Agents to Enhance Immunization
WO2016116907A1 (fr) 2015-01-23 2016-07-28 Helix Biopharma Corporation Conjugués anticorps-uréase pour des fins thérapeutiques
WO2016156537A1 (fr) 2015-03-31 2016-10-06 Novimmune Sa Procédé d'optimisation de l'assemblage et de la production de complexes protéiques hétéro-multimères
WO2017005389A1 (fr) 2015-07-09 2017-01-12 BSH Hausgeräte GmbH Lave-vaisselle pourvu d'un système de sechage
WO2017055389A1 (fr) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Molécules bispécifiques de liaison à l'antigène activant les lymphocytes t anti-ceaxcd3
WO2017118675A1 (fr) 2016-01-08 2017-07-13 F. Hoffmann-La Roche Ag Procédés permettant de traiter des cancers positifs à cea au moyen d'antagonistes de liaison à l'axe de pd-1 et d'anticorps bispécifiques anti-cea/anti-cd3
WO2017118657A1 (fr) 2016-01-05 2017-07-13 Bja Trading Ltd Système de fixation et procédé d'utilisation
EP3199552A1 (fr) 2012-11-20 2017-08-02 Sanofi Anticorps anti-ceacam5 et leurs utilisations
WO2017196793A1 (fr) 2016-05-09 2017-11-16 Celgene Corporation Anticorps cd47 et leurs méthodes d'utilisation
WO2018057955A1 (fr) * 2016-09-23 2018-03-29 Elstar Therapeutics, Inc. Molécules d'anticorps multispécifiques comprenant des chaînes légères lambda et kappa
WO2018098384A1 (fr) * 2016-11-22 2018-05-31 Regents Of The University Of California Modulation par ségrégation pour immunothérapie
WO2018215835A1 (fr) * 2017-05-26 2018-11-29 Novimmune Sa Anticorps anti-cd47 x anti-mésothéline et procédés d'utilisation correspondants
WO2019016411A1 (fr) * 2017-07-21 2019-01-24 Novimmune Sa Génération de mélanges d'anticorps multispécifiques et leurs procédés d'utilisation

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220156667A (ko) 2013-01-10 2022-11-25 젠맵 비. 브이 인간 IgG1 Fc 영역 변이체 및 그의 용도
GB201619652D0 (en) * 2016-11-21 2017-01-04 Alligator Bioscience Ab Novel polypeptides
AU2019236865A1 (en) * 2018-03-23 2020-10-01 Bristol-Myers Squibb Company Antibodies against MICA and/or MICB and uses thereof
WO2019234576A1 (fr) * 2018-06-03 2019-12-12 Lamkap Bio Beta Ltd. Anticorps bispécifiques dirigés contre ceacam5 et cd47

Patent Citations (111)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999054342A1 (fr) 1998-04-20 1999-10-28 Pablo Umana Modification par glycosylation d'anticorps aux fins d'amelioration de la cytotoxicite cellulaire dependant des anticorps
US6602684B1 (en) 1998-04-20 2003-08-05 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
WO2000041474A2 (fr) 1999-01-15 2000-07-20 Novartis Ag Immunotoxines anti-cd3 et leurs utilisations therapeutiques
EP1176195A1 (fr) 1999-04-09 2002-01-30 Kyowa Hakko Kogyo Co., Ltd. Methode de regulation de l'activite d'une molecule immunologiquement fonctionnelle
US20030115614A1 (en) 2000-10-06 2003-06-19 Yutaka Kanda Antibody composition-producing cell
US8067232B2 (en) 2000-10-06 2011-11-29 Kyowa Hakko Kirin Co., Ltd Antibody composition-producing cell with inactivated A-1,6-fusocyltransferase
US6946292B2 (en) 2000-10-06 2005-09-20 Kyowa Hakko Kogyo Co., Ltd. Cells producing antibody compositions with increased antibody dependent cytotoxic activity
US7425446B2 (en) 2000-10-06 2008-09-16 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-producing cell
US20020165360A1 (en) 2000-11-30 2002-11-07 Junghans Richard P. Chimeric effector cell receptors against carcinoembryonic antigen
US20030175884A1 (en) 2001-08-03 2003-09-18 Pablo Umana Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
WO2003056914A1 (fr) 2001-12-27 2003-07-17 Glycofi, Inc. Procede d'ingenierie de structures de carbohydrates de type mammalien
US20040132140A1 (en) 2002-04-09 2004-07-08 Kyowa Hakko Kogyo Co., Ltd. Production process for antibody composition
WO2003085119A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Procede d'amelioration de l'activite d'une composition d'anticorps de liaison avec le recepteur fc$g(g) iiia
US20040110282A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost
WO2003084570A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Medicament contenant une composition d'anticorps appropriee au patient souffrant de polymorphisme fc$g(g)riiia
US20040110704A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells of which genome is modified
WO2003099196A2 (fr) 2002-05-23 2003-12-04 Cure Tech Ltd. Anticorps monoclonaux humanises immunomodulateurs servant a traiter une maladie neoplasique ou une immunodeficience
US20050147614A1 (en) 2002-07-01 2005-07-07 Begent Richard J.H. Antibodies against tumor surface antigens
WO2004024927A1 (fr) 2002-09-12 2004-03-25 Greenovation Biotech Gmbh Procede de production de proteines
WO2004057002A2 (fr) 2002-12-20 2004-07-08 Greenovation Biotech Gmbh Ameliorations apportees ou associees a la production de proteines
US7521051B2 (en) 2002-12-23 2009-04-21 Medimmune Limited Methods of upmodulating adaptive immune response using anti-PD-1 antibodies
WO2004063351A2 (fr) 2003-01-09 2004-07-29 Macrogenics, Inc. Identification et elaboration d'anticorps avec des regions du variant fc et procedes d'utilisation associes
WO2004065540A2 (fr) 2003-01-22 2004-08-05 Glycart Biotechnology Ag Constructions hybrides et leur utilisation pour produire des anticorps presentant une affinite de liaison accrue pour le recepteur fc et fonction d'effecteur
US20040241817A1 (en) 2003-01-22 2004-12-02 Glycart Biotechnology Ag Fusion constructs and use of same to produce antibodies with increased Fc receptor binding affinity and effector function
WO2004099249A2 (fr) 2003-05-02 2004-11-18 Xencor, Inc. Variants fc optimises et leurs procedes de generation
WO2005018669A1 (fr) 2003-08-18 2005-03-03 Macrogenics, Inc. Anticorps specifiques des proteines fc?riib et procedes d'utilisation associes
WO2005018572A2 (fr) 2003-08-22 2005-03-03 Biogen Idec Ma Inc. Anticorps ameliores possedant une fonction d'effecteur modifiee et procedes de fabrication associes
WO2005063815A2 (fr) 2003-11-12 2005-07-14 Biogen Idec Ma Inc. Variants de polypeptides de liaison au recepteur fc$g(g) et procede apparentes
WO2005056759A2 (fr) 2003-12-04 2005-06-23 Xencor, Inc. Procedes pour produire des proteines variantes presentant une plus grande concentration en chaine hote et compositions de celles-ci
WO2005092925A2 (fr) 2004-03-24 2005-10-06 Xencor, Inc. Variantes d'immunoglobuline a l'exterieur de la region fc
WO2005110474A2 (fr) 2004-05-10 2005-11-24 Macrogenics, Inc. ANTICORPS SPÉCIFIQUES FcϜRIIB HUMANISÉS ET MÉTHODES D’UTILISATION
WO2006085967A2 (fr) 2004-07-09 2006-08-17 Xencor, Inc. Anticorps monoclonaux optimises anti-cd20 a variants fc
WO2006019447A1 (fr) 2004-07-15 2006-02-23 Xencor, Inc. Variantes genetiques de fc optimisees
WO2006023420A2 (fr) 2004-08-16 2006-03-02 Medimmune, Inc. Antagonistes de l'integrine possedant une action cytotoxique a mediation cellulaire dependante de l'anticorps amelioree
WO2006047350A2 (fr) 2004-10-21 2006-05-04 Xencor, Inc. Variants d'immunoglobuline igg a fonction effectrice optimisee
WO2007024249A2 (fr) 2004-11-10 2007-03-01 Macrogenics, Inc. Fonction effectrice obtenue par creation par genie biologique de regions d'anticorps fc
WO2006105338A2 (fr) 2005-03-31 2006-10-05 Xencor, Inc. Variants fc presentant des proprietes optimisees
WO2006116260A2 (fr) 2005-04-26 2006-11-02 Medimmune, Inc. Modulation de la fonction d'effecteur d'anticorps par ingenierie de domaine 'charniere'
US8008449B2 (en) 2005-05-09 2011-08-30 Medarex, Inc. Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics
WO2007005874A2 (fr) 2005-07-01 2007-01-11 Medarex, Inc. Anticorps monoclonaux humains diriges contre un ligand de mort programmee de type 1(pd-l1)
US8383796B2 (en) 2005-07-01 2013-02-26 Medarex, Inc. Nucleic acids encoding monoclonal antibodies to programmed death ligand 1 (PD-L1)
WO2007008943A2 (fr) 2005-07-08 2007-01-18 Xencor, Inc. Proteines optimisees qui ciblent la molecule ep-cam
WO2007021841A2 (fr) 2005-08-10 2007-02-22 Macrogenics, Inc. Identification et ingenierie d'anticorps presentant des zones de variants fc et procedes d'utilisation de ces anticorps
WO2007041635A2 (fr) 2005-10-03 2007-04-12 Xencor, Inc. Variants de fc dotés de propriétés de liaison aux récepteurs fc optimisées
WO2007044616A2 (fr) 2005-10-06 2007-04-19 Xencor, Inc. Anticorps anti-cd30 optimises
WO2007042261A2 (fr) 2005-10-11 2007-04-19 Micromet Ag Compositions comportant des anticorps specifiques d'especes croisees et leurs utilisations
US8236308B2 (en) 2005-10-11 2012-08-07 Micromet Ag Composition comprising cross-species-specific antibodies and uses thereof
WO2007048077A2 (fr) 2005-10-21 2007-04-26 Gtc Biotherapeutics, Inc. Anticorps ayant une meilleure activite cytotoxique cellulaire dependant des anticorps et leurs procedes de production et leur utilisation
WO2007071426A1 (fr) 2005-12-21 2007-06-28 Micromet Ag Préparations pharmaceutiques résistantes à l’ace soluble
WO2007106707A2 (fr) 2006-03-10 2007-09-20 Macrogenics, Inc. Identification et modification génétique d'anticorps avec des chaînes lourdes de variants et leurs procédés d'utilisation
WO2008015671A2 (fr) 2006-07-30 2008-02-07 Assaf Klar Procédé de conception et d'application d'une canalisation muticouche à flexibilité longitudinale améliorée
WO2008022152A2 (fr) 2006-08-14 2008-02-21 Xencor, Inc. Anticorps optimisés ciblant cd19
WO2008036688A2 (fr) 2006-09-18 2008-03-27 Xencor, Inc. Anticorps optimisés ciblant l'antigène hm1.24
WO2008121160A2 (fr) 2006-11-21 2008-10-09 Xencor, Inc. Anticorps optimisés qui ciblent cd5
WO2008140603A2 (fr) 2006-12-08 2008-11-20 Macrogenics, Inc. MÉTHODES POUR LE TRAITEMENT DE MALADIE AU MOYEN D'IMMUNOGLOBULINES COMPRENANT DES RÉGIONS FC QUI PRÉSENTENT DES AFFINITÉS ALTÉRÉES POUR FCγR D'ACTIVATION ET FCγR D'INHIBITION
WO2008091798A2 (fr) 2007-01-22 2008-07-31 Xencor, Inc. Anticorps de ca9 optimises et methodes d'utilisation associees
WO2008091954A2 (fr) 2007-01-23 2008-07-31 Xencor, Inc. Anticorps cd40 optimisés et leurs procédés d'utilisation
WO2008092117A2 (fr) 2007-01-25 2008-07-31 Xencor, Inc. Nouvelles insertions, délétions et substitutions d'immunoglobulines
WO2008098115A2 (fr) 2007-02-07 2008-08-14 Xencor, Inc. Anti-corps igf-1r optimisés et procédés utilisant ceux-ci
WO2008119567A2 (fr) 2007-04-03 2008-10-09 Micromet Ag Domaine de liaison spécifique d'espèces croisées
WO2008119566A2 (fr) 2007-04-03 2008-10-09 Micromet Ag Éléments de liaison bispécifiques spécifiques d'espèces croisées
WO2008119565A2 (fr) 2007-04-03 2008-10-09 Micromet Ag Domaine de liaison spécifique d'espèces croisées
WO2008150494A1 (fr) 2007-05-30 2008-12-11 Xencor, Inc. Procédés et compositions permettant l'inhibition de cellules d'expression du cd32b
WO2008156712A1 (fr) 2007-06-18 2008-12-24 N. V. Organon Anticorps dirigés contre le récepteur humain de mort programmée pd-1
US8354509B2 (en) 2007-06-18 2013-01-15 Msd Oss B.V. Antibodies to human programmed death receptor PD-1
WO2009101611A1 (fr) 2008-02-11 2009-08-20 Curetech Ltd. Anticorps monoclonaux pour le traitement de tumeurs
WO2009114335A2 (fr) 2008-03-12 2009-09-17 Merck & Co., Inc. Protéines de liaison avec pd-1
EP2282773A2 (fr) 2008-05-02 2011-02-16 Seattle Genetics, Inc. Procédé et compositions pour préparer des anticorps et des dérivés d'anticorps avec une fucosylation centrale réduite
WO2010027423A2 (fr) 2008-08-25 2010-03-11 Amplimmune, Inc. Compositions d'antagonistes de pd-1 et methodes d'utilisation associees
WO2010027827A2 (fr) 2008-08-25 2010-03-11 Amplimmune, Inc. Polypeptides co-stimulateurs ciblés et leurs procédés d'utilisation dans le traitement du cancer
WO2010027828A2 (fr) 2008-08-25 2010-03-11 Amplimmune, Inc. Antagonistes de pd-1 et leurs procédés d'utilisation
WO2010033736A1 (fr) 2008-09-17 2010-03-25 Xencor, Inc. Nouvelles compositions et nouvelles méthodes de traitement d'affections médiées par les ige
WO2010037836A2 (fr) 2008-10-01 2010-04-08 Micromet Ag Anticorps monocaténaire bispécifique psmaxcd3, spécifique d'espèces croisées
WO2010037838A2 (fr) 2008-10-01 2010-04-08 Micromet Ag Anticorps monocaténaire bispécifique à domaine unique, spécifique d'espèces croisées
WO2010037837A2 (fr) 2008-10-01 2010-04-08 Micromet Ag Anticorps monocaténaires bispécifiques spécifiques d'antigènes cibles de masse moléculaire élevée
WO2010077634A1 (fr) 2008-12-09 2010-07-08 Genentech, Inc. Anticorps anti-pd-l1 et leur utilisation pour améliorer la fonction des lymphocytes t
US20110064653A1 (en) 2009-09-16 2011-03-17 Immunomedics, Inc. Class I Anti-CEA Antibodies and Uses Thereof
US8642292B2 (en) 2009-09-22 2014-02-04 Probiogen Ag Process for producing molecules containing specialized glycan structures
US20120184716A1 (en) 2010-08-16 2012-07-19 Novlmmune S.A. Methods for the Generation of Multispecific and Multivalent Antibodies
WO2012023053A2 (fr) 2010-08-16 2012-02-23 Novimmune S.A. Procédé de production d'anticorps multispécifiques et multivalents
WO2012117002A1 (fr) 2011-03-02 2012-09-07 Roche Glycart Ag Anticorps anti-cea
US20120251529A1 (en) 2011-03-02 2012-10-04 Roche Glycart Ag Anti-cea antibodies
EP2681244B1 (fr) 2011-03-02 2017-11-29 Roche Glycart AG Anticorps anti-cea
WO2013012414A1 (fr) 2011-07-18 2013-01-24 Medimmune, Llc Régimes posologiques pour le traitement cancers exprimant cea
WO2013019906A1 (fr) 2011-08-01 2013-02-07 Genentech, Inc. Procédés de traitement du cancer à l'aide d'antagonistes se liant à l'axe pd-1 et inhibiteurs de mek
WO2013088259A2 (fr) 2011-10-19 2013-06-20 Novimmune S.A. Procédés de purification d'anticorps
US20140140989A1 (en) 2012-02-06 2014-05-22 Inhibrx Llc Non-Platelet Depleting and Non-Red Blood Cell Depleting CD47 Antibodies and Methods of Use Thereof
WO2013173223A1 (fr) 2012-05-15 2013-11-21 Bristol-Myers Squibb Company Immunothérapie anticancéreuse par rupture de la signalisation pd-1/pd-l1
EP3199552A1 (fr) 2012-11-20 2017-08-02 Sanofi Anticorps anti-ceacam5 et leurs utilisations
US20140303354A1 (en) 2012-12-03 2014-10-09 Novimmune S.A. Anti-CD47 Antibodies and Methods of Use Thereof
WO2014087248A2 (fr) 2012-12-03 2014-06-12 Novimmune S.A. Anticorps anti-cd47 et leurs procédés d'utilisation
WO2014113510A1 (fr) 2013-01-15 2014-07-24 Xencor, Inc. Elimination rapide de complexes antigéniques à l'aide de nouveaux anticorps
US20140242079A1 (en) 2013-02-26 2014-08-28 Roche Glycart Ag Bispecific t cell activating antigen binding molecules
US20160144009A1 (en) 2013-04-29 2016-05-26 The Board Of Trustees Of The Leland Stanford Junior University Use of Anti-CD47 Agents to Enhance Immunization
WO2015026634A1 (fr) 2013-08-20 2015-02-26 Merck Sharp & Dohme Corp. Traitement du cancer avec une combinaison d'un antagoniste de pd-1 et du dinaciclib
WO2015112534A2 (fr) 2014-01-21 2015-07-30 Medimmune, Llc Compositions et procédés pour moduler et réorienter des réponses immunitaires
WO2016007235A1 (fr) 2014-07-11 2016-01-14 Genentech, Inc. Anticorps anti-pd-l1 et leurs utilisations
WO2016036678A1 (fr) 2014-09-02 2016-03-10 Medimmune, Llc Formulations d'anticorps bispécifiques
WO2016116907A1 (fr) 2015-01-23 2016-07-28 Helix Biopharma Corporation Conjugués anticorps-uréase pour des fins thérapeutiques
WO2016156537A1 (fr) 2015-03-31 2016-10-06 Novimmune Sa Procédé d'optimisation de l'assemblage et de la production de complexes protéiques hétéro-multimères
WO2017005389A1 (fr) 2015-07-09 2017-01-12 BSH Hausgeräte GmbH Lave-vaisselle pourvu d'un système de sechage
WO2017055389A1 (fr) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Molécules bispécifiques de liaison à l'antigène activant les lymphocytes t anti-ceaxcd3
WO2017118657A1 (fr) 2016-01-05 2017-07-13 Bja Trading Ltd Système de fixation et procédé d'utilisation
WO2017118675A1 (fr) 2016-01-08 2017-07-13 F. Hoffmann-La Roche Ag Procédés permettant de traiter des cancers positifs à cea au moyen d'antagonistes de liaison à l'axe de pd-1 et d'anticorps bispécifiques anti-cea/anti-cd3
WO2017196793A1 (fr) 2016-05-09 2017-11-16 Celgene Corporation Anticorps cd47 et leurs méthodes d'utilisation
WO2018057955A1 (fr) * 2016-09-23 2018-03-29 Elstar Therapeutics, Inc. Molécules d'anticorps multispécifiques comprenant des chaînes légères lambda et kappa
WO2018098384A1 (fr) * 2016-11-22 2018-05-31 Regents Of The University Of California Modulation par ségrégation pour immunothérapie
WO2018215835A1 (fr) * 2017-05-26 2018-11-29 Novimmune Sa Anticorps anti-cd47 x anti-mésothéline et procédés d'utilisation correspondants
WO2019016411A1 (fr) * 2017-07-21 2019-01-24 Novimmune Sa Génération de mélanges d'anticorps multispécifiques et leurs procédés d'utilisation

Non-Patent Citations (56)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 1980
"UniProtKB", Database accession no. FOJ3S3
ANASETTI ET AL., TRANSPLANTATION, vol. 54, 1992, pages 844
BACAC ET AL., CLIN. CANCER RES., vol. 22, no. 13, 2016, pages 3286 - 97
BERINSTEIN N. L., J CLIN ONCOL., vol. 20, 2002, pages 2197 - 2207
BLUMENTHAL RD, BMC CANCER, vol. 7, 3 January 2007 (2007-01-03), pages 2
BRUHNS P., BLOOD, vol. 113, 2009, pages 3716
BRUHNS P., BLOOD, vol. 119, 2012, pages 5640
CHOTHIA ET AL., J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CONAGHHAN P. J. ET AL., BR. J. CANCER, vol. 98, 2008, pages 1217 - 1225
DAVIS J. ET AL., BIOTECHNOL. BIOENG., vol. 74, 2001, pages 288 - 294
DHEILLY E. ET AL., MOL. THERA., vol. 25, 2017, pages 523 - 533
DURBIN H. ET AL., PROC. NATL. SCAD. SCI. USA, vol. 91, 1994, pages 4313 - 4317
GOLDFREEDMAN, J EXP. MED., vol. 121, 1965, pages 439 - 462
HAMMARSTROM S., SEMIN CANCER BIOL., vol. 9, no. 2, 1999, pages 67 - 81
HAO C.ZHANG G., PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE, 2019
HAYASHI, DNA SEQ, vol. 11, 2000, pages 91 - 96
HOHENBERGER, ANNALS SURGERY, 1994
HUANG Y ET AL., J THORAC DIS, vol. 9, no. 2, 2017, pages E168 - E174
J.TABERNERO, J. CLIN. ONCOL., vol. 35, 2017
JOHNSON, G.WU, T.T., NUCLEIC ACIDS RES., vol. 28, 2000, pages 214 - 218
JURGENSMERIER ET AL., BR. J. CANCER, 2013
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1983, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH
KABAT, E.A. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 72, 1975, pages 2785 - 2788
KANDA Y ET AL., J. BIOTECHNOL., vol. 130, 2007, pages 300 - 310
KLEIN ET AL., ONCOIMMUNOLOGY, vol. 6, no. 3, 11 January 2017 (2017-01-11)
KUESPERT K, CURRENT OPINION IN CELL BIOL, vol. 18, 2006, pages 565 - 571
L. CASSARDF. JOENSSONS. ARNAUDM. DAERON, J. IMMUNOL., vol. 189, 2012, pages 2995 - 3006
LUND ET AL., J. IMMUNOL., vol. 157, 1996, pages 4963 - 69
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745
MORI K ET AL., BIOTECHNOL. BIOENG., vol. 88, 2004, pages 901 - 908
NAOKO YAMANE-OHNUKI ET AL., BIOTECH. BIOENG., vol. 87, 2004, pages 614 - 622
NIWA R ET AL., CANCER RES, vol. 64, 2004, pages 2127 - 33
OKAZAKI, A. ET AL., J MOL. BIOL., vol. 336, 2004, pages 1239 - 1249
PEARSON, GENOMICS, vol. 46, 1997, pages 24 - 36
PICCIONE EC ET AL., MABS, vol. 7, 2015, pages 946 - 956
RAMIREZ-SOLIS ET AL., ANAL BIOCHEM, vol. 201, 1992, pages 331 - 335
RICHARDS JO ET AL., MOL. CANCER THER., vol. 7, 2008, pages 2517 - 2527
RICHMAN P. I.BODMER W. F., INT. J. CANCER, vol. 39, 1987, pages 317 - 328
RILLAHAN ET AL., NATURE CHEM. BIOL., vol. 8, no. 7, July 2012 (2012-07-01), pages 661 - 8
SABA JA ET AL., ANAL. BIOCHEM., vol. 305, 2002, pages 16 - 31
SALMERON ET AL., J. IMMUNOL., vol. 147, 1991, pages 3047
SANDLER B. ET AL., ANTICANCER RES, vol. 19, no. 5B, 1999, pages 4229 - 33
SHIELDS, R. L. ET AL., J BIOL. CHEM., vol. 277, 2002, pages 26733 - 26740
STEWART ET AL., CANCER IMMUNOL IMMUNOTHER, vol. 47, 1999, pages 299 - 06
TABERNERO ET AL., J CLIN ONCOL, vol. 35, 2017
UMANA, P. ET AL., NATURE BIOTECHNOL., vol. 17, 1999, pages 176 - 180
VAUGHAN ET AL., NAT. BIOTECH., vol. 14, 1996, pages 309 - 314
VON BOMMEL PE ET AL., ONCOIMMUNOL., vol. 7, 2018, pages e386361
W. R. PEARSON: "Effective protein sequence comparison", METH. ENZYMOL., vol. 266, 1996, pages 227 - 258
W. R. PEARSOND. J. LIPMAN: "Improved Tools for Biological Sequence Analysis", PNAS, vol. 85, 1988, pages 2444 - 2448
WANEBO, NEW ENG. J. MED., 1978
WEISKOPF K., EUROPEAN JOURNAL OF CANCER, vol. 76, 2017, pages 100 - 109
WORMALD ET AL., BIOCHEMISTRY, vol. 36, 1997, pages 1370 - 1380
XU, EXPERT REVIEW OF ANTICANCER THERAPY, vol. 17, 2017, pages 1099 - 1106
YANG SJ, THE JOURNAL OF IMMUNOLOGY, vol. 137, 1986, pages 1097 - 1100

Cited By (2)

* Cited by examiner, † Cited by third party
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US11753481B2 (en) 2020-12-18 2023-09-12 Lamkap Bio Beta Ltd Bispecific antibodies against CEACAM5 and CD47
WO2023186112A1 (fr) * 2022-04-02 2023-10-05 普米斯生物技术(珠海)有限公司 Anticorps multispécifique ciblant ceacam et cd3 et son utilisation

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